Methods of reducing transplant rejection and cardiac allograft vasculopathy by implanting autologous stem cells

ABSTRACT

The invention provides novel methods of reducing transplant rejection and cardiac allograft vasculopathy in humans by employing the implantation of autologous progenitor cells into the transplanted donor heart. The autologous progenitor cells can be vascular progenitor cells (VPCs) and/or myocyte progenitor cells (MPCs) isolated from the recipient&#39;s explanted heart. Alternatively, bone marrow progenitor cells (BMPCs) isolated from the recipient may also be used.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/325,373, filed Dec. 1, 2008, now U.S. Pat. No. 8,124,071, whichclaims the benefit of U.S. Provisional Application No. 60/991,499, filedNov. 30, 2007, both of which are herein incorporated by reference intheir entireties.

FIELD OF THE INVENTION

The present invention relates generally to the field of cardiology, andmore particularly relates to methods of reducing transplant rejection byimplanting cardiac stem cells or bone marrow progenitor cells isolatedfrom the recipient into the donor heart.

BACKGROUND OF THE INVENTION

The interaction between donor and recipient cells after transplantationhas received great attention in an attempt to identify the basis ofrejection and graft-versus-host disease (1-5). Cell migration from theallograft to the recipient results in systemic chimerism (6-8), and cellmigration from the host to the transplanted organ results in chimerismof the organ (9, 10). Chimerism can be detected easily aftersex-mismatched organ transplantation with FISH for the Y-chromosome(11-13) and several studies on cardiac chimerism have providedconsistent results concerning the migration of progenitor cells (PCs)from the host to the graft (14-26). The sex-mismatched transplants makeit possible to document and evaluate quantitatively a process that ispart of cardiac homeostasis but it is otherwise not measurable in humans(27). After homing, host PCs undergo replication and differentiation,generating mature myocytes and vascular structures in the transplantedheart (14). Although there is little disagreement among authors in termsof the occurrence of this phenomenon, the magnitude of cardiac chimerismvaries significantly in different reports (14-27). This discrepancyinvolves mostly ventricular myocytes and to a much lesser extentcoronary vessels. For myocytes, the published values range from as highas 18% (14) to as low as 0.02% (15). Conversely, levels of endothelialcell chimerism and vessel formation have been shown to involve up to 22%of the coronary circulation (14, 25). In spite of these differenceswhich previously have been discussed (27-31), the regeneratedcardiomyocytes and coronary arterioles together with capillary profileshave normal morphology and are distributed predominantly in areas ofintact donor myocardium (14). These data provide evidence that adult PCscontribute to the formation of solid-organ tissue cells (32-36), butleave unanswered the question whether the migrating cells arise fromprecursors in the atrial remnants of the recipient's heart ortranslocate from the recipient's bone marrow through the circulation tothe transplanted organ (37).

After the first year, chronic loss of graft function (71) is thepredominant cause of mortality in cardiac transplant patients (72).Inflammation and immune-mediated reactions (73) are responsible for thereduced sensitivity of myocytes to catecholamines, alterations insurface receptors, defects in ion-channels and depressed contractility(74, 75). Graft dysfunction is characterized by changes in the coronaryarteries by a process termed cardiac allograft vasculopathy (CAV) (76).Although a causal relationship between reduced graft function and CAVremains to be demonstrated, the progressive occlusion of coronaryvessels and ischemic myocardial damage are the critical mechanisms ofgraft failure (77-81). Several risk factors for CAV have beenidentified; they include systemic hypertension, body mass index,advanced donor age and number of rejection episodes (82-90). CAVinexorably leads to a chronic ischemic myopathy and death (91); 75% oftransplant patients suffer from CAV one year following surgery (92, 93).Histologically, four etiologic factors have been considered: (a) intimalthickening mediated by migration of smooth muscle cells and/orproliferation of resident or migratory smooth muscle cells (94, 95); (b)infiltration of the intima by leukocytes recruited in response to injuryor inflammation (96, 97); (c) accumulation of T lymphocytes andmacrophages which generate a peri-vascular cuffing, local injury andirreversible damage, commonly defined as constrictive vascularremodeling (98-103); and (d) dynamic reduction in vessel diametersustained by abnormalities in vasoconstriction and dilation (104-107).CAV differs from typical atherosclerotic lesions (108). With CAV,lesions are concentric and diffuse rather than eccentric and focal andextend beyond the large arteries reaching the penetrating smallerramifications. Because of its diffuse distribution, CAV cannot becorrected with bypass surgery, angioplasty or stenting (72). In somecases, both types of lesions are present.

The cause of graft coronary artery disease remains elusive althoughimmune and non-immune mechanisms have been implicated (70, 109).Controversy exists as to the origin of the proliferating cells presentin CAV (110). It has been proposed that thickening of the intima isdictated by accumulation of recipient cells which derive from a pool ofcirculating PCs that differentiate locally into endothelial cells (ECs)and smooth muscle cells (SMCs) (111). Endothelial progenitor cells(EPCs) from the recipient may home to the intima and differentiate intoendothelial-like cells (112-118) contributing to the vascular lesion.Similarly, SMC precursors could be recruited from the circulation andparticipate in vessel pathology (119-124). The results of cardiacchimerism in humans, however, question the negative effects of PCs ofrecipient origin (14, 15, 19, 20, 23, 25). There is general agreementthat these cells contribute minimally to CAV and the formed coronaryvasculature is structurally intact with no signs of atherosclerosis. Theopposite view is supported by animal studies in which the orthotopicaorta allograft has been employed (117, 125, 126). This model haslimitations; the aorta is structurally different from the coronaryarteries and its intramural branches (127-130). Most importantly, medialnecrosis is present in the orthotopic aorta allograft (125, 126,131-133) while it is never observed in human CAV. These differencesraise questions on the appropriateness of this model for graft vasculardisease. At present, a few effective pharmacological therapies have beenapplied to the treatment of CAV. The HMG-CoA reductase inhibitors andthe cell cycle inhibitor rapamycin reduce neointimal proliferation,myocardial infarction, the need for revascularization and death (134,135). These therapies are extremely valuable but only delay theprogression of CAV in the transplanted heart. Cell therapy with theformation of coronary vessels (47, 48, 51-53, 56, 57, 59, 64, 65) mayincrease coronary blood flow (CBF), decrease coronary resistance andenhance tissue oxygenation (136). The problem in need of resolutioninvolves the identification and characterization of PCs that can formlarge conductive coronary arteries and their distal branches togetherwith a large quantity of cardiomyocytes (59, 64, 137).

SUMMARY OF THE INVENTION

The present invention provides a novel approach to reduce transplantrejection and cardiac allograft vasculopathy in humans. The inventorshave discovered distinct classes of progenitor cells that createimmunocompatible myocardium within the non-immunocompatible transplantedheart and improve myocardial performance, reduce morbidity-mortality andultimately prolong life.

In some embodiments, the methods of the instant invention comprisedelivery of cardiac progenitor cells isolated from the recipient intothe transplanted donor heart, wherein the progenitor cells engraft anddifferentiate into myocytes, smooth muscle cells, and endothelial cellsresulting in the formation of functionally-competent, immunocompatiblemyocardium and coronary vessels. Thus, the present invention provides amethod of reducing an immune response to a transplanted donor heart in asubject. In one embodiment, the method comprises obtaining myocardialtissue from the subject's explanted heart; extracting cardiac progenitorcells from said myocardial tissue; expanding said cardiac progenitorcells in culture; and administering said cardiac progenitor cells to thetransplanted donor heart, wherein said cardiac progenitor cells generateimmunocompatible myocardium and immunocompatible myocardial vesselsfollowing their administration, thereby reducing the immune response tosaid transplanted donor heart. In another embodiment, the cardiacprogenitor cells are separated into vascular progenitor cells andmyocyte progenitor cells prior to administration. Vascular progenitorcells may be c-kit positive and flk1 positive, and differentiate intoimmunocompatible smooth muscle cells and endothelial cells. Myocyteprogenitor cells may be c-kit positive and flk1 negative anddifferentiate into immunocompatible cardiomyocytes. In anotherembodiment, the method further comprises activating the cardiacprogenitor cells prior to administration by exposing the cells to one ormore cytokines.

In another embodiment of the invention, the method of reducing an immuneresponse to a transplanted donor heart in a subject comprises obtaininga bone marrow specimen from the subject; extracting adult bone marrowprogenitor cells from said specimen; expanding said bone marrowprogenitor cells in culture; and administering said bone marrowprogenitor cells to the transplanted donor heart, wherein said bonemarrow progenitor cells generate immunocompatible myocardium andimmunocompatible myocardial vessels following their administration,thereby reducing the immune response to said transplanted donor heart.In some embodiments, the bone marrow progenitor cells are c-kitpositive. In another embodiment, the bone marrow progenitor cells areadministered immediately after transplantation. In another embodiment,the bone marrow progenitor cells differentiate into immunocompatibleendothelial cells, smooth muscle cells, and cardiomyocytes.

In another embodiment of the invention, the method further comprisesextracting cardiac progenitor cells from said subject's explanted heart;separating said cardiac progenitor cells into vascular progenitor cellsand myocyte progenitor cells; and administering said vascular progenitorcells and myocyte progenitor cells to the transplanted donor heart. Thevascular progenitor cells and myocyte progenitor cells may beadministered by multiple administrations after transplantation. Themultiple administrations may occur at a set interval afteradministration of the bone marrow progenitor cells.

In another embodiment of the invention, the method of reducing an immuneresponse to a transplanted donor heart in a subject comprises obtainingmyocardial tissue from the subject's explanted heart; extracting cardiacmyocyte progenitor cells from said myocardial tissue; expanding saidmyocyte progenitor cells in culture; and administering said myocyteprogenitor cells to the transplanted donor heart, wherein said myocyteprogenitor cells generate immunocompatible myocardium following theiradministration, thereby reducing the immune response to saidtransplanted donor heart. The myocyte progenitor cells may be c-kitpositive and flk1 negative. In one embodiment, the myocyte progenitorcells differentiate into immunocompatible cardiomyocytes.

The present invention also provides a method of reducing cardiacallograft vasculopathy in a subject who has received a transplanteddonor heart. In one embodiment, the method comprises obtainingmyocardial tissue from the subject's explanted heart; extracting cardiacvascular progenitor cells from said myocardial tissue; expanding saidvascular progenitor cells in culture; and administering said vascularprogenitor cells to the transplanted donor heart, wherein said vascularprogenitor cells generate immunocompatible coronary vasculature, therebyrepairing/and or regenerating the non-immunocompatible coronary arteriesof the donor heart. The vascular progenitor cells may be c-kit positiveand flk1 positive. In one embodiment, the vascular progenitor cellsdifferentiate into immunocompatible endothelial cells and smooth musclecells.

In some embodiments, the methods described herein may further compriseadministering to the subject an immunosuppressive therapy. Theimmunosuppressive therapy may be administered concurrently orsubsequently to the administration of the progenitor cells to the donorheart. Preferably, the dose and frequency of a standardimmunosuppressive therapy is reduced following the administration of oneor more types of progenitor cells (e.g. bone marrow progenitor cells,vascular progenitor cells, or myocyte progenitor cells) to the donorheart.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Administration of progenitor cells (PCs) to the cardiactransplant. Bone marrow is harvested from the recipient male dog andmononuclear cells are lineage depleted and sorted for c-kit (BMPCs).Following transplantation of a female donor heart, the explanted heartfrom the recipient is dissociated and vascular progenitor cells (VPCs)and myocyte progenitor cells (MPCs) are isolated and expanded. PCclasses will be infected with a lentivirus expressing EGFP, β-gal or RFPfor the subsequent identification of the injected cells and theirprogeny at different time points. Newly formed EGFP-, β-gal andRFP-positive myocardial structures will develop within the donormyocardium. Since labeled-PCs are given repeatedly over time, thecoronary route is considered the most feasible form of cell delivery.

FIG. 2. Cardiac chimerism. A 67-year-old man died 9 days after sexmismatched heart transplant. The donor female heart showed regenerationfoci characterized by clusters of developing myocytes. A-D: α-SA: red;nuclei: DAPI, blue. Myocyte boundaries were defined by laminin (yellow).The analysis of sex-chromosomes by FISH documented the male genotype ofthe new myocytes (arrows) which carried at most one Y-chr (green-dots)and one X-chr (magenta-dots). Large myocytes (asterisks) had a femalegenotype (two X-chr). A: one new myocyte contained two sets of X- andY-chr suggesting cell fusion (double arrows). However, phospho-H3(inset, white) demonstrated mitotic division and excluded cell fusion.Similar examples were found in other transplanted hearts.

FIG. 3. Chimerism of the dog heart. Donor female heart transplanted inmale recipient. Subsequently the donor heart was injected withautologous-recipient EGFP-positive PCs. Several small developingmyocytes are present (A, B: α-SA: red). These new myocytes are alsoEGFP-positive (B: α-SA-EGFP, yellow). Three of these cells are shown athigher magnification in the inset (B). Analysis of sex-chromosomesdocumented the male genotype of the forming myocytes (arrows) whichcarried at most one Y-chr (white dots) and one X-chr (magenta dots).Female recipient myocytes show at most two X-chr (*).

FIG. 4. Transplanted dog heart. A, B: Areas of myocardial damageoccupied by inflammatory cells. C: These cells are CD4 (B lymphocytes)and CD8 (T lymphocytes) positive. D-F: Lymphocytic infiltrates in theluminal and abluminal aspects of a large and intermediate branch of LAD.

FIG. 5. Dog heart: vascular and myocardial niches. A: Resistancecoronary arteriole; c-kit-positive cells (green) are present in theintima (vWf, yellow, *), SMC layer (α-SMA, red, arrows) and adventitia(not stained, arrowheads). B: The c-kit-positive-cells express flk1(white), i.e., VPC niche. C, D: Tangential section of myocardialcapillaries that exhibit 3 c-kit-positive flk1-positive VPCs. Connexin43 (yellow, arrows) is present between VPCs and ECs. E: Large section ofa dog coronary artery (SMCs, α-SMA; red). Area in the rectangle is shownin panel F: 2 VPCs are connected to SMCs. The inset illustrates connexin43 (yellow, arrows). (G, H: LV myocardium which shows a cluster ofc-kit-positive flk1-negative cells (green), i.e., MPC niche. Connexin 43(arrowheads) and N-cadherin (arrows) are present between two MPCs, andbetween MPCs and myocytes (α-SA, red) or MPCs and fibroblasts(procollagen, light blue).

FIG. 6. PC classes. Freshly isolated dog BMPCs, and VPCs and MPCsexpanded in vitro (P3-P4). A: BMPCs were stained with antibodies forblood lineages. B: Lineage negative c-kit-positive BMPCs were identifiedand sorted and analyzed in cytospin preparations; c-kit (green). C: VPCsare positive for c-kit and flk1 and negative for hematopoietic markersand α-SA; they express at very low levels desmin, CD31, vWf and TGF-β1receptor. D: VPCs are c-kit-positive (green) and flk1-positive (red). E:MPCs are negative for flk1 and for hematopoietic markers, CD31, vWf andTGF-β1 receptor; they express at very low levels α-SA and desmin. F:MPCs are c-kit-positive (green) and flk1-negative.

FIG. 7. Clones and derived progeny. Single VPCs isolated from dogcoronary arteries (A) and MPCs from the myocardium (B) formedmulticellular clones. VPCs are c-kit (green) and flk1 (red) positive.MPCs are c-kit (green) positive and flk1-negative. C: Clonogenic VPCsdifferentiate into SMCs (α-SMA, green), 59-4%, ECs (vWf, yellow), 31±4%,and myocytes (α-SA, red), 10±2%. D: Clonogenic MPCs differentiate intoSMCs, 16±6%, ECs, 11±5%, and myocytes, 73±9%.

FIG. 8. Translocation of PCs. Migrating EGFP-MPCs (A & B) are locatedwithin interstitial fibronectin tunnels (yellow). Arrows point to thedirection of migration of the EGFP-MPCs established in living tissue bytwo-photon microscopy.

FIG. 9. PC translocation. A: Two-photon microscopy: 20 min afterinjection, EGFP-MPCs (green) are within the lumen of coronary vessels(red). B-F: Transcoronary migration of EGFP-MPCs; images of the samefield were taken at 30 min intervals. Arrowheads point to 2 EGFP-MPCsdetected in the living tissue. G: After fixation, migrated cells wereidentified by confocal microscopy. Myocytes are stained by cardiacmyosin heavy chain (MHC: magenta).

FIG. 10. Position of the catheter (A, C) and injection of cardiac PCstogether with Isopaque into the circumflex coronary artery (B) and theLAD (D) of a transplanted dog. Echocardiographic images of thetransplanted heart at 8 (E) and 15 (F) days after surgery. (G) Isopaqueand cell viability.

FIG. 11. Transplanted heart. A: The heart was cut in 22 slices and 114sections were examined. B: Large transverse section of the LV in which acluster of EGFP-positive (green) myocytes (α-sarcomeric actin, α-SA:red) is located in proximity of three areas of myocardial damage (MD).More than 2000 EGFP-positive cells are present. C: Four other examplesof clusters of EGFP-positive myocytes within the transplanted heart. D:Newly formed EGFP-positive coronary vessels (EGFP: green; α-smoothmuscle actin, α-SA: red). Inset: co-localization of EGFP and α-SMA inthe vessel wall (yellow). E: Higher magnification of newly formedmyocytes (left panels: EGFP, green; right panels: α-SA: red; arrows). F:EGFP-positive cells express GATA4 (red), Nkx2.5 (red) and GATA6 (red)documenting the acquisition of the myocyte and SMC lineage. G, H: Newlyformed myocytes (α-SA, red) carry the Y-chr (white dots). I: By PCR,EGFP DNA sequences were detected only in the heart (H) but not in thekidney (K), spleen (S), lung (Lu) and liver (L). J: The recognition ofSry by PCR in the heart (H) indicates the presence of male cells ofrecipient origin in the donor heart. K, S, Lu and L correspond to organsof the recipient dog that have therefore a male genotype (SEQ ID NOS.: 3and 4).

FIG. 12. BMPC differentiation and cell genotyping. A: Transgeneconstructs in donor mice. The promoter that controls the ubiquitous ormyocyte restricted expression of the transgene is shown. Also the schemeillustrates that male donor BMPCs were injected intramyocardially inwild-type female infarcted mice. NLS, nuclear localization signal; MHC,myosin heavy chain; MI, myocardial infarct. Three classes of BMPCs wereemployed to induce myocardial regeneration: 1. Male EGFP-positive BMPCsfrom β-actin-EGFP mice; 2. Male EGFP-negative BMPCs from α-MHC-EGFPmice; and 3. Male EGFP-negative BMPCs from α-MHC-c-myc-tagged-nuc-Aktmice. B: In case 1, all cardiac cells generated by BMPC differentiationare expected to express EGFP; in case 2, only myocytes generated by BMPCdifferentiation are expected to express EGFP; and in case 3, only nucleiof myocytes generated by BMPC differentiation are expected to expressc-myc-tag. C: The detection of the Y-chr allowed us to discriminateresident female cardiac cells from newly formed male cardiac cellsgenerated by BMPC differentiation.

FIG. 13. A: BMPCs from α-MHC-c-myc-tagged-nuc-Akt mice regeneratedmyocytes (α-SA: red; c-myc: green; arrowheads). B: Infarcts treated withBMPCs from β-actin-EGFP mice. EP: epicardium; EN: endocardium. Left,central and right panels show EGFP (green), new myocytes (MHC, red) andtheir merge. Arrows: non regenerated infarct. C: Left, central and rightpanels illustrate new myocytes (MHC, red), distribution of Y-chr (white)and their merge. Arrows: non regenerated infarct. D: Highermagnification of new myocytes within the formed myocardium. The restoredmyocyte mass increased with time. E: DNA sequences (see SEQ ID NOS.:5-10) of EGFP and c-myc-tag by PCR. DNA from the tail of donor TG and WTmice was employed as + and − control. F: Transcripts and sequences (seeSEQ ID NOS.: 11-16) for EGFP and c-myc-tag by RT-PCR in infarctedtreated hearts (+). Absence of RT reaction (−). RNA from hearts of TGand WT was employed as + and − control. G: EGFP and c-myc-tag protein byWestern blotting.

FIG. 14. Lack of cell fusion. Regenerated human myocytes (A) and vessels(B) are Cre-recombinase positive (white) but EGFP-negative. C: Positivecontrol: EGFP-myocytes of mice in which EGFP was driven by α-MHCpromoter. D: Myocytes and vessels show at most two human X-chr (whitedots; arrowheads). Mouse X-Chr (magenta dots; arrows) are present inmyocytes bordering the infarct (BZ). See ref 139 for detail.

FIG. 15. PC engraftment. Male BMPCs (Y-chr: white dots) injected in theinfarcted female mouse heart express N-cadherin (A: yellow) and connexin43 (B: yellow). Connexin 43 and N-cadherin were detected between BMPCsand between BMPCs and resident myocytes (α-SA: red) and fibroblasts(procollagen: magenta). Apoptosis of BMPCs was restricted tonon-engrafted cells (C); connexin 43 was absent in TdT labeled PCs(magenta). Engrafted BMPCs are BrdU-positive (D: yellow). Mitosis isshown by phospho-H3 labeling of metaphase chromosomes (E: yellow).

FIG. 16. A: A doppler flow transducer and hydraulic occluder wereimplanted on the LAD. A critical stenosis was created as shown by theabsence of reactive hyperemia after release (R) of 15-sec occlusion. Inthe absence of stenosis (B), the release leads to reactive hyperemiashown as mean and phasic CBF in the upper and lower. C: Lack of reactivehyperemnia with critical stenosis 10 days after LAD constriction and theinjection of EGFP-positive VPCs in six sites around the stenotic artery(A: red stars). As documented in D, 30 days after coronary constrictionand cell implantation there was a slow return of CBF after release of15-sec occlusion. These data suggest the formation of coronary vesselswhich restored in part CBF in the presence of a functional criticalstenosis. Histologically, at 30 day, in proximity of the stenoticvessel, a large developing artery (E: diameter=˜1 mm) was identified(EGFP: green; α-SMA: red; arrowheads). Preexisting vessels are EGFPnegative and α-SMA positive (arrows).

FIG. 17. AKANE protocol (Amplification of the Key DNA sequence Adjacentto an integrated provirus by Nested PCR coupled with Enzymatic digestionand ligation of the genome). This method corresponds to an inverse PCRwhich is the most sensitive strategy for the amplification of unknownDNA sequences that flank a region of known sequence. The primers areoriented in the reverse direction of the usual orientation and thetemplate for the reverse primers is a restriction fragment that has beenligated and self-circularized. A: The AKANE method was first developedin human cardiac PCs infected with an EGFP lentivirus in vitro at lowefficiency (˜10%). Genomic DNA was extracted, digested with EcoR1 andligated with T4 DNA ligase to circularize the DNA fragments. Two sets ofPCR primers were used to amrplify the coding region of the EGFP gene:(1) one set of conventional primers that produced a single band of 164bp and (2) a set of primers with opposite directions that amplify onlycircularized DNA and produced a single band of 143 bp. B: To determinethe clonal profile of the infected human cardiac PCs, extracted DNA wasdigested with Taq1, ligated with T4 DNA ligase and re-linearized withHind3. One round of PCR and two additional nested PCR were perfbormed.The PCR primers employed in the first (1st) and second (2nd)amplification round were designed in the region of Long Terminal Repeat,LTR, which is commonly located at the 5′- and 3′-sides of the lentiviralgenome. The PCR primers employed in the third round (3rd) were specificfor either the 5′- or the 3′-side of the site of integration. In allcases, primers were oriented in the opposite direction. No clear bandswere detected after the 1st round because of the low amount of thetarget templates in the sample. Arrows of the same color point to thecorresponding bands in different lanes. As expected, the size of theamplicons decreased by 112 bp for the 5′-side products and 138 bp fobthe 3′-side products. The seven bands indicated by the arrows reflectdifferent sites of integration of the EGFP lentivirus in the genomic DNAof the infected human cardiac PCs. Two of the amplified bands wereexcised from the gel and the DNA was re-amplified as shown in the lasttwo lanes. C: DNA sequencing analysis of the two re-amplified bandsdemonstrated that the DNA contained the proviral genome, shown in green,together with the key DNA sequence of the human genome, shown in blue.The human genome is linked to the proviral DNA through the integrationsite and the Taq1 restriction site. The sites of integration werelocated in chromosomes 20 and 6, respectively. D: Multiple clones wereidentified in human cardiac PCs, and the identified chromosomes arelisted. E: After EGFP lentiviral infection (˜80% efficiency), humancardiac PCs were implanted in infarcted rats that were sacrificed onemonth later. Cardiac cells were enzymatically digested and separated incardiac PCs, ECs, fibroblasts and myocytes. By the AKANE protocol,various clones were identified: one clone in c-kit positive cells, eightclones in ECs, four clones in fibroblasts, and six clones in myocytes.F: DNA sequencing analysis demonstrated that the DNA contained theproviral genome (green) together with the key DNA sequence of the humangenome (blue). One of the detected clones was present in all committedcell types indicating that these cells constitute the progeny of one ofthe injected c-kit positive cardiac PCs which underwent multi lineagedifferentiation. G: An example of the detected sites of integration inthe various cardiac cells of the same animal are listed. Some cloneswere common to different category of the cells (arrowheads). Thedistribution of the integration sites, again, confirmed the randomintegration of the EGFP lentivirus into the human genome.

FIG. 18. A: Angiogenesis corresponds to the sprouting of mature ECs andSMCs from pre-existing vessels. Therefore, the cells within the newlyformed vessel will not express the reporter genes carried by theinjected PCs. B: Vasculogenesis corresponds to sites of activeneovascularization and is mediated by the recruitment of the deliveredPCs. In this case, the cells in the regenerated vessels will express thereporter genes. C: Alternatively, Vasculogenesis corresponds to therecruitment of circulating EPCs and smooth muscle PCs (SMPCs), whichtogether generate new vessels. In this case, the cells in the vesselwall will be negative for the reporter genes but will carry the Y-chr.DI: Adaptive arteriogenesis or collateral vessel formation corTespondsto the development of large vessels from pre-existing arteriolaranastomoses. Therefore, the cells within the wall of the collateralvessels will be negative for the reporter genes. E: Combination ofangiogenesis and vasculogenesis may also occur resulting in thecolocalization of cells negative and positive for the reporter geneswithin the wall of newly formed vessels.

FIG. 19. Physiology of newly generated myocytes. A: Contractility,action potential, calcium transients and voltage clamp currents in asmall newly formed (left panel) and a large terminally differentiated(right panel) myocyte. Hypertrophied myocytes exhibit depressedcontractility, which is paralleled by shorter action potential,decreased calcium transient and increased net outward current. B:Mechanical properties of myocytes generated by BMPCs injected in theinfarcted mouse heart. Newly formed cells show green fluorescence, sinceBMPCs were obtained from transgenic mice expressing EGFP under thecontrol of α-MHC promoter. Spared hypertrophied myocytes (phasecontrast) are characterized by decreased fractional shortening. C:Membrane currents in myocytes generated by BMPCs. In comparison withsurviving myocytes, new cells possess higher outward potassium currentwhich, in turn, determines longer duration of the action potential.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, “autologous” refers to something that is derived ortransferred from the same individual's body (i.e., autologous blooddonation; an autologous bone marrow transplant).

As used herein, “allogeneic” refers to something that is geneticallydifferent although belonging to or obtained from the same species (e.g.,allogeneic tissue grafts or organ transplants).

As used herein, “stem cells” are used interchangeably with “progenitorcells” and refer to cells that have the ability to renew themselvesthrough mitosis as well as differentiate into various specialized celltypes. The stem cells used in the invention are somatic stem cells, suchas bone marrow or cardiac stem cells or progenitor cells. “Vascularprogenitor cells” or VPCs are a subset of adult cardiac stem cells thatare c-kit positive and flk1 positive, which generate predominantlyendothelial cells and smooth muscle cells. “Myocyte progenitor cells” orMPCs are a subset of adult cardiac stem cells that are c-kit positiveand flk1 negative, which generate cardiomyocytes predominantly.

As used herein, “adult” stem cells refers to stem cells that are notembryonic in origin nor derived from embryos or fetal tissue.

Stem cells (e.g. progenitor cells) employed in the invention areadvantageously selected to be lineage negative. The term “lineagenegative” is known to one skilled in the art as meaning the cell doesnot express antigens characteristic of specific cell lineages. Forexample, bone marrow progenitor cells (BMPCs) do not express any of thehematopoietic lineage markers, such as CD3, CD20, CD33, CD14, and CD15.And, it is advantageous that the lineage negative stem cells areselected to be c-kit positive. The term “c-kit” is known to one skilledin the art as being a receptor which is known to be present on thesurface of stem cells, and which is routinely utilized in the process ofidentifying and separating stem cells from other surrounding cells.

As used herein, the term “immunocompatible” refers to the antigenicsimilarity of cells or tissues from a donor source to the cells ortissues in a recipient subject such that the donor cells or tissues donot induce an immune response in the recipient subject. A donor cell ortissue that does not induce an immune response in a recipient and is notrejected by the recipient subject is said to be immunocompatible.

As used herein, the term “cytokine” is used interchangeably with “growthfactor” and refers to peptides or proteins that bind receptors on cellsurfaces and initiate signaling cascades thus influencing cellularprocesses. The terms “cytokine” and “growth factor” encompass functionalvariants of the native cytokine or growth factor. A functional variantof the cytokine or growth factor would retain the ability to activateits corresponding receptor. Variants can include amino acidsubstitutions, insertions, deletions, alternative splice variants, orfragments of the native protein. The term “variant” with respect to apolypeptide refers to an amino acid sequence that is altered by one ormore amino acids with respect to a reference sequence. The variant canhave “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties, e.g., replacement of leucinewith isoleucine. Alternatively, a variant can have “nonconservative”changes, e.g., replacement of a glycine with a tryptophan. Analogousminor variations can also include amino acid deletion or insertion, orboth. Guidance in determining which amino acid residues can besubstituted, inserted, or deleted without eliminating biologicalactivity can be found using computer programs well known in the art, forexample, DNASTAR software.

As used herein, “patient” or “subject” may encompass any vertebrateincluding but not limited to humans, mammals, reptiles, amphibians andfish. However, advantageously, the patient or subject is a mammal suchas a human, or a mammal such as a domesticated mammal, e.g., dog, cat,horse, and the like, or production mammal, e.g., cow, sheep, pig, andthe like.

The pharmaceutical compositions of the present invention may be used astherapeutic agents—i.e. in therapy applications. As herein, the terms“treatment” and “therapy” include curative effects, alleviation effects,and prophylactic effects. In certain embodiments, a therapeuticallyeffective dose of progenitor cells is applied, delivered, oradministered to the heart or implanted into the heart. An effective doseor amount is an amount sufficient to effect a beneficial or desiredclinical result. Said dose could be administered in one or moreadministrations.

Mention is made of the following related pending patent applications:

U.S. Application Publication No. 2003/0054973, filed Jun. 5, 2002, whichis herein incorporated by reference in its entirety, discloses methods,compositions, and kits for repairing damaged myocardium and/ormyocardial cells including the administration of cytokines.

U.S. Application Publication No. 2006/0239983, filed Feb. 16, 2006,which is herein incorporated by reference in its entirety, disclosesmethods, compositions, and kits for repairing damaged myocardium and/ormyocardial cells including the administration of cytokines and/or adultstem cells as well as methods and compositions for the development oflarge arteries and vessels. The application also discloses methods andmedia fbr the growth, expansion, and activation of human cardiac stemcells.

Successful transplantation has an inherent yearly 6% mortality and mostpatients die within 10-12 years. The improved immunosuppressive regimenhas resulted in increased short-term survival but long-term survival hasremained largely the same. Post-operative morbidity and mortalityassociated with cardiac transplantation is dictated by acute and chroniccomplications which rapidly affect the performance of the new heart orlead with time to a progressive deterioration of cardiac function.Chronic rejection with accelerated cardiac allograft vasculopathy (CAV)is the major pathological event that determines the fatal evolution ofthe transplanted heart. Graft failure due to CAV is characterized byocclusive vessel disease that results in myocardial infarction, multiplefocal areas of tissue injury, arrhythmias, sudden death and congestiveheart failure. Infarct healing with scar formation is impaired byimmunosuppressive therapy which further complicates the devastatingconsequences of graft atherosclerosis and arteriosclerosis. Themechanisms of graft atherosclerosis and arteriosclerosis are notunderstood but repetitive cell-mediated and/or humoral-mediatedimmunological damage followed by a potentiated myointimal response areinvolved in the manifestations of this unique form of coronary arterydisease. Thus, three interrelated pathologic processes appear to becritical determinants of heart failure in the transplanted heart:rejection, evolving coronary atherosclerosis and chronic ischemicmyocardial injury.

Chronic rejection with accelerated graft atherosclerosis is the majorpathological event that determines the fatal evolution of thetransplanted heart. Occlusive vessel disease of the large, intermediateand small coronary arteries results in multiple focal areas of injury,myocardial infarction and ischemic heart failure. The novel methods ofthe invention provide a solution to the major problems associated withthe unfavorable progression of cardiac transplantation in humans byemploying progenitor cells from the recipient to repopulate the donorheart with immunocompatible cardiomyocytes and coronary vessels.

Functionally competent cardiac progenitor cells also known as cardiacstem cells are present in the explanted heart of patients undergoingcardiac transplantation and these can be isolated and grown in vitro forsubsequent autologous cell therapy. Additionally, in the acute phase,bone marrow progenitor cells (BMPCs) from the recipient can be utilizedin view of their ability to transdifferentiate and acquire thecardiomyocyte fate and form vascular structures.

Thus, the donor heart can provide the scaffolding for the generation ofa new heart derived from implantation, engraftment and differentiationof BMPCs and cardiac progenitor cells isolated from the recipient. Twodistinct classes of cardiac progenitor cells exist within the heart:vascular progenitor cells (VPCs) and myocyte progenitor cells (MNPCs).VPCs are programmed to differentiate predominantly into vascular smoothmuscle cells (SMCs) and endothelial cells (ECs), but also have theability to acquire the cardiomyocyte lineage. On the other hand, MPCspredominantly generate cardiomyocytes and to a more limited extent SMCsand ECs.

Successful engraftment of progenitor cells is the initial process oftissue repair. Myocardial reconstitution necessitates the generation ofa cardiomyocyte compartment together with a proportional coronaryvasculature. Myocytes alone in the absence of adequate blood supplycannot perform their function, and coronary vessels alone without musclemass cannot restore cardiac performance (36, 184, 185). Engraftedprogenitor cells may result in a coordinated growth response in whichmyocytes and vessels are concurrently formed to engender functionallycompetent myocardium. Coronary blood flow is regulated by conductivecoronary arteries and resistance coronary arterioles (186) while oxygenavailability and diffusion are controlled by the capillary network (136,188).

BMPCs, VPCs, and MPCs isolated from the recipient can be delivered tothe donor heart to promote the formation of immunocompatible myocardiumwithin the non-immunocompatible transplanted heart. The reconstitutedmyocardium will be comprised of immunocompatible coronary vessels andcoronary myocytes which can replace and/or repair thenon-immunocompatible diseased arteries and myocardium. Thus,immunosuppressive therapy may no longer be required resulting insignificant improvement of quality of life and lifespan of patientsafter cardiac transplantation.

The present invention provides methods of reducing transplant rejectionin a subject by isolating cardiac or bone marrow progenitor cells fromtissue specimens from the recipient, expanding and optionally activatingthe progenitor cells in culture, and subsequently administering therecipient's progenitor cells to the transplanted donor heart. Theimplanted progenitor cells then generate immunocompatible endothelialcells, smooth muscle cells, and cardiomyocytes within thenon-immunocompatible donor myocardium, which assemble intoimmunocompatible myocardium and myocardial vessels, thus reducing theimmune response to the donor organ.

In one embodiment, the present invention provides a method of reducingan immune response to a transplanted donor heart in a subject comprisingobtaining myocardial tissue from the subject's explanted heart;extracting cardiac progenitor cells from said myocardial tissue;expanding said cardiac progenitor cells in culture; and administeringsaid cardiac progenitor cells to the transplanted donor heart, whereinsaid cardiac progenitor cells generate immunocompatible myocardium andimmunocompatible myocardial vessels following their administration,thereby reducing the immune response to said transplanted donor heart.In some embodiments, the subject is human.

It is preferable that the cardiac progenitor cells be c-kit positive.The c-kit stem cell marker is associated with progenitor cells withapparent comparable functional behavior for cardiac repair whether theyderive from the heart or the bone marrow (47, 48, 51, 57, 59, 64, 139,140). In some embodiments, the cardiac progenitor cells are separatedinto vascular progenitor cells and myocyte progenitor cells prior toadministration. Recently, the inventors have discovered that the adultheart in mice, dogs and humans contains two populations of progenitorcells (PCs); a vascular progenitor cell (VPC), which is stored in nicheslocated within the wall of coronary vessels, and a myocyte progenitorcell (MPC), which is located in myocardial niches, distinct fromvascular niches (FIG. 5). See also U.S. Provisional Application No.60/991,515, which is herein incorporated by reference in its entirety.VPCs are primitive cells which are self-renewing, clonogenic andmultipotent in vitro and can regenerate coronary vessels in vivo.Although coronary VPCs are programmed to differentiate predominantlyinto smooth muscle cells (SMCs) and endothelial cells (ECs), they alsopossess the inherent ability to acquire modestly the cardiomyocytelineage. Conversely, MPCs generate predominantly cardiomyocytes and to alimited extent vascular SMCs and ECs.

The c-kit marker is present in the absence of flk1 in MPCs (29, 64,138-140) and together with flk1 in VPCs (FIG. 5). Therefore, the pool oflineage negative c-kit-positive PCs in the heart may be separated intotwo cell categories according to the expression of the vascularendothelial growth factor receptor 2 (VEGF-R2/flk1). The expression ofVEGFR2 or kinase domain receptor (KDR/flk1), which represents theearliest marker of angioblast precursors (141-145), is a good predictorof VPCs. Flk1 is an epicardium-specific marker and epicardial-derivedcells initiate vasculogenesis in the prenatal heart (146, 147). In themouse embryo, flk1 is necessary for the development of the coronaryvasculature (148, 149). Recent results have suggested that the growthpotential of flk1-positive PCs exceeds hematopoiesis and vasculogenesis(144, 145, 150-156). The endocardium and a small population of cells inthe myocardium originate from a pool of flk1-positive cells (153-156).Additionally, multipotent flk1 PCs form colonies of ECs, SMCs andmyocytes (145). Thus, the expression of flk1 and c-kit may be utilizedto distinguish VPCs and MPCs. In one embodiment, the vascular progenitorcells are c-kit positive and flk1 positive. In another embodiment, thevascular progenitor cells differentiate into immunocompatibleendothelial cells and smooth muscle cells. In another embodiment, themyocyte progenitor cells are c-kit positive and flk1 negative. In stillanother embodiment, the myocyte progenitor cells differentiate intoimmunocompatible cardiomyocytes.

In another embodiment, the present invention provides a method ofreducing an immune response to a transplanted donor heart in a subjectcomprising obtaining myocardial tissue from the subject's explantedheart; extracting cardiac myocyte progenitor cells from said myocardialtissue; expanding said myocyte progenitor cells in culture; andadministering said myocyte progenitor cells to the transplanted donorheart, wherein said myocyte progenitor cells generate immunocompatiblemyocardium following their administration, thereby reducing the immuneresponse to said transplanted donor heart. In some embodiments, themyocyte progenitor cells are c-kit positive and flk1 negative. In otherembodiments the myocyte progenitor cells differentiate intoimmunocompatible cardiomyocytes.

Adult bone marrow progenitor cells (BMPCs) are capable of generatingmature cells beyond their own tissue boundaries, a process which hasbeen termed developmental plasticity. Based on this premise, theinventors have documented that BMPCs regenerate infarcted myocardium inrodents leading to the formation of cardiomyocytes and coronary vesselswhich are structurally and functionally connected to residentcardiomyocytes and the primary coronary circulation. Thus, BMPCs canalso be employed to generate immunocompatible myocardium to preventrejection of a transplanted heart. Because BMPCs can be obtained fromthe recipient subject prior to the transplant surgery, the BMPCs can beexpanded in culture and ready for administration to the donor heart atthe time of the transplant surgery. Therefore, in another embodiment ofthe invention, the method of reducing an immune response to atransplanted donor heart in a subject comprises obtaining a bone marrowspecimen from the subject; extracting adult bone marrow progenitor cellsfrom said specimen; expanding said bone marrow progenitor cells inculture; and administering said bone marrow progenitor cells to thetransplanted donor heart, wherein said bone marrow progenitor cellsgenerate immunocompatible myocardium and immunocompatible myocardialvessels following their administration, thereby reducing the immuneresponse to said transplanted donor heart. In some embodiments, the bonemarrow progenitor cells are administered immediately aftertransplantation.

The stem cell antigen c-kit is expressed in a population of BMPCs thatare capable of differentiating into cardiomyocytes and SMCs and ECsorganized in coronary vessels (47, 48, 51). Therefore, the presence ofc-kit may be employed to isolate BMPCs. Accordingly, in one embodimentof the invention, the bone marrow progenitor cells are c-kit positive.In another embodiment, the bone marrow progenitor cells differentiateinto immunocompatible endothelial cells, smooth muscle cells, andcardiomyocytes.

In another embodiment, vascular progenitor cells and/or myocyteprogenitor cells may be administered to the donor heart followingadministration of bone marrow progenitor cells. For instance, in someembodiments, the method further comprises extracting cardiac progenitorcells from said subject's explanted heart; separating said cardiacprogenitor cells into vascular progenitor cells and myocyte progenitorcells; and administering said vascular progenitor cells and myocyteprogenitor cells to the transplanted donor heart. In such embodiments,the BMPCs administered at the time of transplant surgery initiate thegeneration of immunocompatible myocardial tissue and vessels, and thisgeneration is expanded by the subsequent administration of VPCs andMPCs. The VPCs and MPCs may be administered multiple times aftertransplantation and the multiple administrations may occur at a setinterval after the administration of the BMPCs. For example, VPCs and/orMPCs may be administered to the donor heart every week, two weeks, threeweeks, month, two months, three months, six months, nine months, year,two years, three years, or five years after BMPC administration.

The present invention also provides a method of reducing cardiacallograft vasculopathy in a subject who has received a transplanteddonor heart. Cardiac allograft vasculopathy is an accelerated form ofcoronary artery disease that affects the vasculature of the allograftand is the primary cause of death in transplant patients surviving oneyear after transplantation. The non-immunocompatible tissue of theallograft induces an immune response in the recipient subject that leadsto endothelial cell damage and vascular injury. The vascular injuryinitiates a repair response that can lead to the occlusion of the vesseland subsequent infarction.

Use of cardiac progenitor cells isolated from the recipient may be usedto generate immunocompatible myocardial tissue in the donor heart toreduce an immune response, and thus also reduce the development ofcardiac allograft vasculopathy. In addition, vascular progenitor cellsobtained from the recipient subject can be employed to generateimmunocompatible vessels in the donor heart that would not besusceptible to immune-mediated injury. Thus, in one embodiment of theinvention, the method of reducing cardiac allograft vasculopathy in asubject comprises obtaining myocardial tissue from the subject'sexplanted heart; extracting cardiac vascular progenitor cells from saidmyocardial tissue; expanding said vascular progenitor cells in culture;and administering said vascular progenitor cells to the transplanteddonor heart, wherein said vascular progenitor cells generateimmunocompatible coronary vasculature, thereby repairing/and orregenerating the non-immunocompatible coronary arteries of the donorheart. In some embodiments, the vascular progenitor cells are c-kitpositive and flk1 positive. In other embodiments, the vascularprogenitor cells differentiate into immunocompatible endothelial cellsand smooth muscle cells.

Progenitor cells may be isolated from tissue specimens, such asmyocardium or bone marrow, obtained from a subject or patient, such asthe transplant recipient. By way of example, myocardial tissue specimensobtained from the recipient's explanted heart may be minced and placedin appropriate culture medium. Cardiac progenitor cells growing out fromthe tissue specimens can be observed in approximately 1-2 weeks afterinitial culture. At approximately 4 weeks after the initial culture, theexpanded progenitor cells may be collected by centrifugation. Anexemplary method for obtaining bone marrow progenitor cells from asubject is described as follows. Bone marrow may be harvested from theiliac crests using a needle and the red blood cells in the sample may belysed using standard reagents. Bone marrow progenitor cells arecollected from the sample by density gradient centrifugation.Optionally, the bone marrow progenitor cells may be expanded in culture.Other methods of isolating adult progenitor cells, such as bone marrowprogenitor cells and cardiac progenitor cells, from a subject are knownin the art and can be employed to obtain suitable progenitor cells foruse in the methods of the invention. U.S. Patent Application PublicationNo. 2006/0239983, filed Feb. 16, 2006, which is herein incorporated byreference in its entirety, describes media appropriate for culturing andexpanding adult progenitor cells. However, one of ordinary skill in theart would be able to determine the necessary components and modifycommonly used cell culture media to be employed in culturing theisolated progenitor cells of the invention.

It is preferable that the progenitor cells of the invention are lineagenegative. Lineage negative progenitor cells can be isolated by variousmeans, including but not limited to, removing lineage positive cells bycontacting the progenitor cell population with antibodies againstlineage markers and subsequently isolating the antibody-bound cells byusing an anti-immunoglobulin antibody conjugated to magnetic beads and abiomagnet. Alternatively, the antibody-bound lineage positive stem cellsmay be retained on a column containing beads conjugated toanti-immunoglobulin antibodies. For instance, lineage negative bonemarrow progenitor cells may be obtained by incubating mononuclear cellsisolated from a bone marrow specimen with immunomagnetic beadsconjugated with monoclonal antibodies for CD13 (T lymphocytes), CD20 (Blymphocytes), CD33 (myeloid progenitors), CD14 and CD15 (monocytes). Thecells not bound to the immunomagnetic beads represent the lineagenegative bone marrow progenitor cell fraction and may be isolated.Similarly, cells expressing markers of the cardiac lineage may beremoved from cardiac progenitor cell populations to isolate lineagenegative cardiac progenitor cells.

In a preferred embodiment of the invention, the lineage negativeprogenitor cells express the stem cell surface marker, c-kit, which isthe receptor for stem cell factor. Positive selection methods forisolating a population of lineage negative progenitor cells expressingc-kit are well known to the skilled artisan. Examples of possiblemethods include, but are not limited to, various types of cell sorting,such as fluorescence activated cell sorting (FACS) and magnetic cellsorting as well as modified forms of affinity chromatography. In apreferred embodiment, the lineage negative progenitor cells are c-kitpositive. In some embodiments, c-kit positive cardiac progenitor cellsare further separated into subpopulations of cells expressing the VEGFR2receptor, flk1. Cardiac progenitor cells that are c-kit positive andflk1 positive are vascular progenitor cells, while cardiac progenitorcells that are c-kit positive and flk1 negative are myocyte progenitorcells. Similar positive selection methods for isolating c-kit positiveprogenitor cells may be used to select cells expressing the flk1receptor (e.g. immunobeads, cell sorting, affinity chromatography,etc.).

Isolated lineage negative, c-kit positive progenitor cells may be platedindividually in single wells of a cell culture plate and expanded toobtain clones from individual progenitor cells. In some embodiments,cardiac progenitor cells that are c-kit positive and flk1 positive areplated individually to obtain pure cultures of vascular progenitorcells. In other embodiments, cardiac progenitor cells that are c-kitpositive and flk1 negative are plated individually to obtain purecultures of myocyte progenitor cells.

In certain embodiments of the invention, the cardiac progenitor cells orbone marrow progenitor cells are activated prior to administration.Activation of the progenitor cells may be accomplished by exposing theprogenitor cells to one or more cytokines. Suitable concentrations ofthe one or more cytokines for activating the progenitor cells include aconcentration of about 0.1 to about 500 ng/ml, about 10 to about 500ng/ml, about 20 to about 400 ng/ml, about 30 to about 300 ng/ml, about50 to about 200 ng/ml, or about 80 to about 150 ng/ml. In oneembodiment, the concentration of one or more cytokines is about 25,about 50, about 75, about 100, about 125, about 150, about 175, about200, about 225, about 250, about 275, about 300, about 325, about 350,about 375, about 400, about 425, about 450, about 475, or about 500ng/ml. In some embodiments, the cardiac progenitor cells or bone marrowprogenitor cells are activated by contact with hepatocyte growth factor(HGF), insulin-like growth factor-1 (IGF-1), or variant thereof.

HGF positively influences stem cell migration and homing through theactivation of the c-Met receptor (Kollet et al. (2003) J. Clin. Invest.112: 160-169; Linke et al. (2005) Proc. Natl. Acad. Sci. USA 102:8966-8971; Ros-Myles et al. (2005) J. Cell. Sci. 118: 4343-4352; Urbaneket al. (2005) Circ. Res. 97: 663-673). Similarly, IGF-1 and itscorresponding receptor (IGF-IR) induce cardiac stem cell division,upregulate telomerase activity, hinder replicative senescence andpreserve the pool of functionally-competent cardiac stem cells in theheart (Kajstura et al. (2001) Diabetes 50: 1414-1424; Torella et al.(2004) Circ. Res. 94: 514-524; Davis et al. (2006) Proc. Natl. Acad.Sci. USA 103: 8155-8160). In some embodiments, the cardiac progenitorcells or bone marrow progenitor cells are contacted with HGF and IGF-1.

Some other non-limiting examples of cytokines that are suitable for theactivation of the cardiac progenitor cells or bone marrow progenitorcells include Activin A, Bone Morphogenic Protein 2, Bone MorphogenicProtein 4, Bone Morphogenic Protein 6, Cardiotrophin-1, FibroblastGrowth Factor 1, Fibroblast Growth Factor 4, Flt3 Ligand, Glial-DerivedNeurotrophic Factor, Heparin, Insulin-like Growth Factor-II,Insulin-Like Growth Factor Binding Protein-3, Insulin-Like Growth FactorBinding Protein-5, Interleukin-3, Interleukin-6, Interleukin-8, LeukemiaInhibitory Factor, Midkine, Platelet-Derived Growth Factor AA,Platelet-Derived Growth Factor BB, Progesterone, Putrescine, Stem CellFactor, Stromal-Derived Factor-1, Thrombopoietin, Transforming GrowthFactor-α, Transforming Growth Factor-β1, Transforming Growth Factor-β2,Transforming Growth Factor-□β3, Vascular Endothelial Growth Factor,Wnt1, Wnt3a, and Wnt5a, as described in Kanemura et al. (2005) CellTransplant. 14:673-682; Kaplan et al. (2005) Nature 438:750-751; Xu etal. (2005) Methods Mol. Med. 121:189-202; Quinn et al. (2005) MethodsMol. Med. 121:125-148; Almeida et al. (2005) J Biol Chem.280:41342-41351; Barnabe-Heider et al. (2005) Neuron 48:253-265;Madlambayan et al. (2005) Exp Hematol 33:1229-1239; Kamanga-Sollo et al.(2005) Exp Cell Res 311:167-176; Heese et al. (2005) Neuro-oncol.7:476-484; He et al. (2005) Am J Physiol. 289:H968-H972; Beattie et al.(2005) Stem Cells 23:489-495; Sekiya et al. (2005) Cell Tissue Res320:269-276; Weidt (2004) Stem Cells 22:890-896; Encabo et al (2004)Stem Cells 22:725-740; and Buytaeri-Hoefen et al. (2004) Stem Cells22:669-674, the entire text of each of which is incorporated herein byreference.

Functional variants of the above-mentioned cytokines can also beemployed in the invention. Functional cytokine variants would retain theability to bind and activate their corresponding receptors. Variants caninclude amino acid substitutions, insertions, deletions, alternativesplice variants, or fragments of the native protein. For example, NK1and NK2 are natural splice variants of HGF, which are able to bind tothe c-MET receptor. These types of naturally occurring splice variantsas well as engineered variants of the cytokine proteins that retainfunction can be employed to activate the progenitor cells of theinvention.

The present invention involves administering a therapeutically effectivedose or amount of progenitor cells to a donor heart. An effective doseis an amount sufficient to effect a beneficial or desired clinicalresult. Said dose could be administered in one or more administrations.In some embodiments, at least three effective doses are administered tothe donor heart in the recipient subject. In other embodiments, at leastfive effective doses are administered to the donor heart in therecipient subject. Each administration of progenitor cells may comprisea single type of progenitor cell (e.g. BMPC, VPC, or MPC) or may containmixtures of the different types of progenitor cells. In one embodiment,bone marrow progenitor cells (BMPCs) are administered to the subject atthe time of transplantation, and vascular progenitor cells (VPCs) and/ormyocyte progenitor cells (MPCs) are administered at set intervals aftertransplantation. Examples of suitable intervals include, but are notlimited to, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6months, 12 months, 18 months or 24 months.

An effective dose of progenitor cells may be from about 2×10⁴ to about1×10⁷, more preferably about 1×10⁵ to about 6×10⁶, or most preferablyabout 2×10⁶. As illustrated in the examples, 2×10⁶ to 1×10⁷ progenitorcells are used to effect regeneration of immunocompatible myocardium ina canine model. Although there would be a size difference between theheart of a canine and the heart of a human, it is likely that this rangeof progenitor cells would be sufficient in a human as well. However, theprecise determination of what would be considered an effective dose maybe based on factors individual to each patient, including their size,age, size of donor heart, type of repopulating progenitor cells (e.g.VPCs, MPCs, or BMPCs), and amount of time after transplantation. One skilied in the art, specifically a physician or cardiologist, would be ableto determine the number of progenitor cells that would constitute aneffective dose without undue experimentation.

The progenitor cells (e.g. stem cells) may be administered to the heartby injection. The injection is preferably intramyocardial. As oneskilled in the art would be aware, this is the preferred method ofdelivery for stem cells as the heart is a functioning muscle. Injectionby this route ensures that the injected material will not be lost due tothe contracting movements of the heart.

In another embodiment, the progenitor cells are administered byinjection transendocardially or trans-epicardially. In anotherembodiment of the invention, the progenitor cells are administered usinga catheter-based approach to deliver the trans-endocardial injection.The use of a catheter precludes more invasive methods of deliverywherein the opening of the chest cavity would be necessitated. As oneskilled in the art would appreciate, optimum time of recovery would beallowed by the more minimally invasive procedure. A catheter approachinvolves the use of such techniques as the NOGA catheter or similarsystems. The NOGA catheter system facilitates guided administration byproviding electromechanic mapping of the area of interest, as well as aretractable needle that can be used to deliver targeted injections or tobathe a targeted area with a therapeutic. Any of the embodiments of thepresent invention can be administered through the use of such a systemto deliver injections or provide a therapeutic. One of skill in the artwill recognize alternate systems that also provide the ability toprovide targeted treatment through the integration of imaging and acatheter delivery system that can be used with the present invention.Information regarding the use of NOGA and similar systems can be foundin, for example, Sherman (2003) Basic Appl. Myol. 13: 11-14; Patel etal. (2005) The Journal of Thoracic and Cardiovascular Surgery130:1631-38; and Perrin et al. (2003) Circulation 107: 2294-2302; thetext of each of which are incorporated herein in their entirety.

In still another embodiment, the progenitor cells may be administered toa donor heart by an intracoronary route. This route obviates the need toopen the chest cavity to deliver the cells directly to the heart. One ofskill in the art will recognize other useful methods of delivery orimplantation which can be utilized with the present invention, includingthose described in Dawn et al. (2005) Proc. Natl. Acad. Sci. USA 102,3766-3771, the contents of which are incorporated herein in theirentirety.

In some embodiments, the methods of the invention described hereinfurther comprise administering to the subject an immunosuppressivetherapy or imnmunosuppressant. Non-limiting examples ofimmunosuppressants include cyclosporine A, azathioprine, glucocorticoids(e.g. methylprednisolone, cortisol, prednisone, dexamethasone,betamethasone), cyclophosphamide, methotrexate, mercaptopurine,dactinomycin, anthracyclines, mitomycin C, bleomycin, mithramycin,tacrolimus, sirolimus, everolimus, myriocin, and antibodies (e.g.basiliximab, daclizumab, anti-thymocyte globulin, anti-lymphocyteglobulin). Other therapeutic agents that are typically given totransplant patients, for instance HMG-CoA reductase inhibitors,rapamycin, and paclitaxel, may also be used in combination with theadministration of progenitor cells. The immunosuppressants or othertherapeutic agents may be administered to the subject in multiple dosessubsequent to the administration of the progenitor cells. Theimmunosuppressants or other therapeutic agents may be taken on a routineschedule for a set period of time. For example, the immunosuppressantsor other therapeutic agents may be taken once daily for about 1 month,about 2 months, about 3 months, about 6 months, about 12 months, orabout 24 months after transplantation and administration of theprogenitor cells. Other dosing schedules may be employed. Preferably,the dose and/or frequency of immunosuppressants or other therapeuticagents will be reduced following one or more administrations of theprogenitor cells to the donor heart. One of skill in the art,particularly a physician or cardiologist, would be able to determine theappropriate dose and schedule for the administration of theimmunosuppressants or other therapeutic agents.

The invention also comprehends methods for preparing compositions, suchas pharmaceutical compositions, including one or more of the differenttype of progenitor cells described herein, for instance, for use ininventive methods for reducing cardiac allograft vasculopathy ortransplant rejection. In one embodiment, the pharmaceutical compositioncomprises bone marrow progenitor cells and a pharmaceutically acceptablecarrier, wherein said bone marrow progenitor cells are c-kit positive.In another embodiment, the pharmaceutical composition comprises vascularprogenitor cells and a pharmaceutically acceptable carrier, wherein saidvascular progenitor cells are c-kit positive and flk1 positive. Inanother embodiment, the pharmaceutical composition comprises myocyteprogenitor cells and a pharmaceutically acceptable carrier, wherein saidmyocyte progenitor cells are c-kit positive and flk1 negative. In stillanother embodiment, the pharmaceutical composition comprises vascularprogenitor cells, myocyte progenitor cells and a pharmaceuticallyacceptable carrier, wherein said vascular progenitor cells are c-kitpositive and flk1 positive and said myocyte progenitor cells are c-kitpositive and flk1 negative.

In an additionally preferred aspect, the pharmaceutical compositions ofthe present invention are delivered via injection. These routes foradministration (delivery) include, but are not limited to, subcutaneousor parenteral including intravenous, intraarterial (e.g. intracoronary),intramuscular, intraperitoneal, intramyocardial, transendocardial,trans-epicardial, intranasal administration as well as intrathecal, andinfusion techniques. Accordingly, the pharmaceutical composition ispreferably in a form that is suitable for injection.

When administering a therapeutic of the present invention parenterally,it will generally be formulated in a unit dosage injectable form(solution, suspension, emulsion). The pharmaceutical formulationssuitable for injection include sterile aqueous solutions or dispersionsand sterile powders for reconstitution into sterile injectable solutionsor dispersions. The carrier can be a solvent or dispersing mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol, and the like), suitablemixtures thereof and vegetable oils.

Proper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion, and by the use of surfactants. Nonaqueousvehicles such a cottonseed oil, sesame oil, olive oil, soybean oil, cornoil, sunflower oil, or peanut oil and esters, such as isopropylmyristate, may also be used as solvent systems for compoundcompositions.

Additionally, various additives which enhance the stability, sterility,and isotonicity of the compositions, including antimicrobialpreservatives, antioxidants, chelating agents, and buffers, can beadded. Prevention of the action of microorganisms can be ensured byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, and the like. In many cases, it willbe desirable to include isotonic agents, for example, sugars, sodiumchloride, and the like. Prolonged absorption of the injectablepharmaceutical form can be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin. According tothe present invention, however, any vehicle, diluent, or additive usedwould have to be compatible with the progenitor cells and othercompounds used in combination with the progenitor cells.

Sterile injectable solutions can be prepared by incorporating thecompounds utilized in practicing the present invention in the requiredamount of the appropriate solvent with various amounts of the otheringredients, as desired.

The pharmaceutical compositions of the present invention, e.g.,comprising a therapeutic dose of progenitor cells (e.g. BMAPCs, VPC, andMPCs), can be administered to the patient in an injectable formulationcontaining any compatible carrier, such as various vehicles, adjuvants,additives, and diluents. Compounds, such as immunosuppressants or othertherapeutic agents, to be administered as a combination therapy with theprogenitor cells can be administered parenterally to the patient in theform of slow-release subcutaneous implants or targeted delivery systemssuch as monoclonal antibodies, iontophoretic, polymer matrices,liposomes, and microspheres.

Examples of compositions comprising a therapeutic of the inventioninclude liquid preparations for parenteral, subcutaneous, intradermal,intramuscular, intracoronarial, intramyocardial or intravenousadministration (e.g., injectable administration), such as sterilesuspensions or emulsions. Such compositions may be in admixture with asuitable carrier, diluent, or excipient such as sterile water,physiological saline, glucose or the like. The compositions can also belyophilized. The compositions can contain auxiliary substances such aswetting or emulsifying agents, pH buffering agents, gelling or viscosityenhancing additives, preservatives, flavoring agents, colors, and thelike, depending upon the route of administration and the preparationdesired. Standard texts, such as “REMINGTON'S PHARMACEUTICAL SCIENCE”,17th edition, 1985, incorporated herein by reference, may be consultedto prepare suitable preparations, without undue experimentation.

The compositions can be isotonic, i.e., they can have the same osmoticpressure as blood and lacrimal fluid. The desired isotonicity of thecompositions of this invention may be accomplished using sodiumchloride, or other pharmaceutically acceptable agents such as dextrose,boric acid, sodium tartrate, propylene glycol or other inorganic ororganic solutes. Sodium chloride is preferred particularly for bufferscontaining sodium ions.

Viscosity of the compositions may be maintained at the selected levelusing a pharmaceutically acceptable thickening agent. Methylcellulose ispreferred because it is readily and economically available and is easyto work with. Other suitable thickening agents include, for example,xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer,and the like. The preferred concentration of the thickener will dependupon the agent selected. The important point is to use an amount whichwill achieve the selected viscosity. Viscous compositions are normallyprepared from solutions by the addition of such thickening agents.

A pharmaceutically acceptable preservative can be employed to increasethe shelf-life of the compositions. Benzyl alcohol may be suitable,although a variety of preservatives including, for example, parabens,thimerosal, chlorobutanol, or benzalkonium chloride may also beemployed. A suitable concentration of the preservative will be from0.02% to 2% based on the total weight although there may be appreciablevariation depending upon the agent selected.

Those skilled in the art will recognize that the components of thecompositions should be selected to be chemically inert with respect tothe active compound. This will present no problem to those skilled inchemical and pharmaceutical principles, or problems can be readilyavoided by reference to standard texts or by simple experiments (notinvolving undue experimentation), from this disclosure and the documentscited herein.

The inventive compositions of this invention are prepared by mixing theingredients following generally accepted procedures. For example,isolated progenitor cells can be resuspended in an appropriatepharmaceutically acceptable carrier and the mixture adjusted to thefinal concentration and viscosity by the addition of water or thickeningagent and possibly a buffer to control pH or an additional solute tocontrol tonicity. Generally the pH may be from about 3 to 7.5.Compositions can be administered in dosages and by techniques well knownto those skilled in the medical and veterinary arts taking intoconsideration such factors as the age, sex, weight, and condition of theparticular patient, and the composition form used for administration(e.g., liquid). Dosages for humans or other mammals can be determinedwithout undue experimentation by the skilled artisan, from thisdisclosure, the documents cited herein, and the knowledge in the art.

Suitable regimes for initial administration and further doses or forsequential administrations also are variable, may include an initialadministration followed by subsequent administrations; but nonetheless,may be ascertained by the skilled artisan, from this disclosure, thedocuments cited herein, and the knowledge in the art.

This invention is further illustrated by the following additionalexamples that should not be construed as limiting. The contents of allreferences, patents and published patent applications cited throughoutthis application, as well as the Figures, are incorporated herein byreference in their entirety.

EXAMPLES Example 1 Cardiac Transplantation in Dogs

In all examples described herein, female dog hearts are transplanted inmale dogs and the cells to be injected (bone marrow progenitor cells(BMPCs), vascular progenitor cells (VPCs) or myocyte progenitor cells(MPCs)) are infected with a lentivirus expressing enhanced greenfluorescent protein (EGFP), β-gal or red fluorescent protein (RFP) sothat the progeny formed by cells given at different time points can beidentified and measured.

In the last two years, a tremendous effort was made to develop a modelof cardiac transplantation in the dog. The dog model was chosen aftercareful review because the dog is the smallest animal that fits theexperimental requirements. Immunosuppression is well described in dogs(198-200) and canine MPCs and VPCs (64; FIGS. 5-7) have been isolated,expanded and characterized in vitro and in vivo. Thus, the use of thedog model maximizes efficiency.

In addition, data in the transplanted dog heart model show inflammatoryinfiltrates in the major coronary vessels and distal branches togetherwith initial myointimal thickening (FIG. 4). Additionally, vast zones ofdamage characterized by clusters of T′ lymphocytes, monocytes andmacrophages have been detected in the ventricular myocardium. Typically,myocytes are lost and collagen accumulates in the areas of injury. Ofthe 113 samples examined in this case myocardial pathology wasidentified in 90. These results indicate that the transplanted dog heartis a good model of cardiac allograft vasculopathy which occurs in humancardiac transplants.

A. Surgery

The female donor dog is positioned, heparin, 300 IU/kg, administered andanesthesia induced by thiopental, 6-10 mg/kg. A left lateral thoracotomyis performed, the pericardium opened, cardioplegic solution (ViaSpan)infused and the heart explanted and kept on ice. The male recipient dogis given pre-operative analgesia with oral carprofen and fentanyl patch2 hours before surgery. Cephalic vein intravenous access is established.The animal is anesthetized (pre-anesthetics: atropine, 0.04 mg/kg, andtelazol, 4 mg/kg; anesthetic: thiopental, 6-10 mg/kg), injected withheparin and placed in a warming blanket to preserve normothermia.Analgesia during surgery is provided by a bolus injection of 5 μg/kgfentanyl. Throughout surgery, anesthesia is maintained with isoflurane,1-3% in 100% oxygen, delivered via an orotracheal tube. A neuromuscularblocking agent cisatracurium, 0.25 mg/kg, is given after depthanesthesia is established. The ECG is continuously recorded. An oralgastric tube is introduced for decompression and methylprednisolone, 500mg, is given as initial imnmunosuppressive dose. Following thoracotomyand opening of the pericardium, the superior and inferior vena cava arelooped with umbilical tapes and cannulated for venous drainage. Thedescending thoracic aorta is cannulated for arterial return andcardiopulmonary bypass initiated. The dog is maintained at 37-38° C.using a heater cooler within the cardiopulmonary bypass circuit. Theaorta is cross-clamped and the heart removed, leaving a posteriorremnant containing portions of the right and the left atria. The donorheart is sewn in using running, continuous 3-0 and 4-0 prolene suture.The atrial cuffs are joined first; the aorta and pulmonary arteryanastomoses are then connected. The aortic clamp is removed and theheart is de-aired and allowed to recover. After reperfusion (⅓ of thetime of cold ischemic period), the recipient is weaned off theextracorporeal circulation. Anticoagulation is reversed with protamine(1 mg/mg heparin given). Epicardial pacing wires and a chest tube areplaced, thoracotomy closed in layers, air evacuated from the chestcavity and the tube secured to the skin. Vital signs (blood pressure,heart and respiratory rate, urine output and oxygen saturation) aremonitored and recorded every hour for the first 4 hours and every twohours for the subsequent 14 hours. Arterial blood gases and electrolytesare periodically checked until the acidosis has resolved and the bloodcount has stabilized. Dogs remain on the ventilator overnight withNormosol-R intravenous drip, 2 ml/kg/h. On the morning of the firstpostoperative day, dogs that are hemodynamically stable are extubated.

B. Drug Administration

For analgesia, dogs receive a fentanyl transdermal patch, 75 μg/h, andcarprofen, 4.4 mg/kg for 7 days postoperatively. Animals showingdiscomfort are further treated with buprenorphine, 0.02 mg/kg every 12hours. The antibiotic cefazolin, 10 mg/kg is administered every 8 hoursfor 2 days. Beginning the day of transplantation, recipient dogs receivetriple drug immunosuppression consisting of cyclosporine A, 18mg/kg/day, azathioprine, 2 mg/kg/day, and methylprednisolone, 50 mg/day.The trough level of cyclosporine A is determined twice a week andimmunosuppression is adjusted until a stable level of the agent isreached. Aspirin, 81 mg, is given daily to prevent venousthromboembolism.

C. Instrumentation

During transplantation, the recipient dog is chronically instrumented. ATygon catheter is placed into the descending thoracic aorta to measurearterial pressure. Probes are implanted in the donor heart duringextracorporeal circulation. A solid-state pressure gauge (Konigsberg) isinserted in the left ventricle through the apex. A Doppler-flowtransducer is placed around the left circumflex coronary artery tomeasure blood flow and a pair of 3-MHz piezoelectric crystals are fixedon opposing endocardial surfaces at the base of the left ventricle.Wires and catheters are run subcutaneously to the intrascapular region.After recovery, dogs are trained to lie quietly on the laboratory table(64, 201-204).

D. Hemodynamics

Measurements are obtained at 10 days after each cell treatment and atsacrifice. The aortic catheter is connected to a P23 ID strain-gaugetransducer to measure aortic pressure. LV pressure is determined anddP/dt is calculated. LV diameter is measured by connecting the implantedpiezoelectric crystals to a transit time ultrasonic dimension gauge thatgenerates a voltage linearly proportional to the transit time of theultrasound traveling between the two crystals (1.55×μm/s). The analogsignals are digitized at a sampling rate of 500 Hz. Systolic, diastolic,and mean arterial pressures, positive and negative dP/dt, heart rate,end-diastolic and end-systolic diameter, and pressure-diameter loopareas are evaluated (201-204).

E. Echocardiography

In conscious dogs, M-mode recordings are made from short axis views,with 21) guidance. LV chamber dimensions and wall thickness are measuredin a plane below the mitral valve and perpendicular to the LV in anM-mode recording, LV chamber volume is assessed in a two-dimensionalparasternal long axis view. LV volumes are calculated using thehemi-cylindrical hemi-ellipsoid model. Stroke volume is computed as thedifference between LV end-systolic and end-diastolic volume. Cardiacoutput corresponds to the product of stroke volume and heart rate.Myocardial wall stress (WS) is calculated from the product ofintraventricular pressure (LVP), radius of curvature (R) and wallthickness (h): WS=LVP×R/h.

F. Cell Infection

Progenitor cells (PCs) are infected with lentiviruses carrying EGFP(green), β-gal (blue) or RFP (red) so that the contribution of separatecell injections to the transplanted heart can be establishedquantitatively. In a separate set of studies, PCs to be injected aredivided in three equal parts and infected respectively with lentivirusescarrying EGFP-Flag-tag under the cardiomyocyte specific α-MHC promoter,RFP-HA-tag under the SMC-specific Sm22a promoter and TFP-c-myc-tag underthe EC-specific VE-cadherin promoter so that the generation of myocytes,smooth muscle cells (SMCs) and endothelial cells (ECs) in thetransplanted heart are determined quantitatively by real-time RT-PCR andWestern blotting by measuring the expression of the reporter constructsat the mRNA and protein level (57). These biochemical measurements arecomplemented with immunocytochemical determinations.

G. Cell Implantation

The intra-coronary route was selected to deliver the progenitor cells tothe donor heart. This route was chosen to obviate the need for multiplesurvival surgeries that would be required with intramyocardial injectionof cells. Experiments were performed in rats subjected to brief episodesof ischemia followed by reperfusion to document that progenitor cellscross the vessel wall and reach the myocardium (183). Ischemia typicallyoccurs at transplantation (71). Movement of EGFP-tagged MPCs (green) wasassessed ex vivo by two-photon microscopy after perfusion of thecoronary vasculature with rhodamine labeled dextran (red). EGFP-labeledMPCs migrate across the wall of coronary vessels and within 3 hours moveinto the ischemic area (FIG. 9; see ref. 183 for detail). Thus,intra-arterial delivery of MPCs leads to their extravasation and homingto the myocardial interstitium.

In the transplanted dog, left Amplatz or right Judkins catheter isinserted into the left or right coronary artery under fluoroscopy. Cellsare suspended in 0.5-2.0 mL of Isopaque and injected. The dye allows thevisualization of the coronary vasculature and delivery of cells (FIG.10). Then, the catheter is removed and the vessels repaired. Dogs aregiven antibiotics baytril, 6 mg/kg, and trimethoprim/sulfa, 33 mg/kg.Isopaque does not affect cardiac PC viability for as long as 16 hours.During the procedure, cardiac PCs are exposed to Isopaque for less than5 min.

H. Generation of Immunocompatible Myocardium

In these initial studies, VPCs and MPCs were not separated and the PCpool was isolated from the explanted heart and expanded in vitro. Cellswere infected with EGFP lentivirus (65% efficiency). Male EGFP-PCs weregiven 15 and 24 days after transplantation and the donor heart wasexamined 6 days after the second treatment, i.e., 30 days followingsurgery. The heart was sliced in 22 sections ˜4 mm each and severalsamples were obtained from each section (FIG. 11A): 114 samples wereanalyzed histologically. A similar protocol is used in all studiesdiscussed in Examples 2-5. Clusters of newly formed EGFP-positivemyocytes and coronary vessels (FIG. 11B-E) were detected in 109 of 114specimens of the left and right ventricle. The ability of EGFP-PCs todifferentiate into myocytes and SMCs was confirmed by detection ofmyocyte transcription factors, GATA4 and Nkx2.5, and the SMCtranscription factor, GATA6 (FIG. 11F). The detection of the Ychromosome (Y-chr) confirmed the male genotype of the regeneratedmyocytes (FIG. 11G-H). As shown before, at most one X- and Y-chr wereidentified in the formed cells excluding cell fusion (FIG. 3). Incontrast, at most 2 X-chr were seen in donor cardiac cells documentingtheir female genotype. The engraftment and survival of male EGFP-PCs wasconfirmed by PCR for EGFP DNA and the Sry gene located in theY-chromosome (FIG. 11I and J). Random sampling of tissue from kidney,spleen, lung and liver failed to reveal DNA sequences for EGFP by PCR(FIG. 11I). Thus, at 15 and 6 days after the first and second injectionno EGFP-PCs were found to be engrafted outside of the heart. Comparablefindings were obtained in a second dog in which male EGFP-PCs were given15 and 22 days after transplant and the animal was sacrificed 2 weekslater, i.e., 45 days after surgery.

I. Immunohistochemistry with Quantum Dots, FISH and Confocal Microscopy

The recognition of regenerated male myocardium within the female donorheart requires immunolabeling of structures and confocal microscopy. Toevaluate the lineage commitment of differentiating cells, antibodies forthe following proteins are utilized. Markers for myocytes include GATA4,Nkx2.5, MEF2C, α-SA, α-cardiac actinin, troponin I, troponin T, cardiacMHC, atrial and ventricular myosin light chain (MLC), connexin 43 andN-cadherin; for SMCs, GATA-6, TGF-β1 receptor, α-SMA and calponin; andfor ECs, Ets1, Vezf, CD31 and vWf (14, 47, 48, 51, 57, 59, 64, 137,139). Y- and X-chr are identified by FISH (14, 47, 51, 57, 139) withcanine specific probes (Cambio).

Example 2 Implantation of BMPCs from the Recipient into the TransplantedDonor Heart Generates Immunocompatible Coronary Vessels and MnyocytesImproving the Evolution of the Cardiac Graft

This Example demonstrates that bone marrow progenitor cells (BMPCs) canacquire the cardiomyocyte, and vascular smooth muscle cell (SMC) andendothelial cell (EC) lineages in support of the therapeutic efficacy ofBMPCs. Additionally, the consequences of cell fusion events andparacrine effects on myocardial regeneration are addressed.

A. Transdifferentiation

To date, the hematopoietic stem cell appears to be the most versatilestem cell in crossing lineage boundaries and the most prone to break thelaw of tissue fidelity (40, 205). Early studies on BMPC differentiationinto myocardium have generated great enthusiasm (47, 51, 52, 168) butother observations have rejected the initial results (41-43) andpromoted a wave of skepticism about the therapeutic potential of BMPCsfor the injured heart (46, 206). The major criticisms include inaccurateinterpretation of the original data due to autofluorescence artifactsand the lack of genetic markers for the recognition of the donor BMPCsand their progeny (41-43, 46, 206).

To address these issues, female infarcted mice were injected with maleBMPCs obtained from transgenic mice in which EGFP was under the controlof the ubiquitous β-actin promoter and the consequences of thisintervention on post-infarction remodeling were determined. In anotherset of experiments donor BMPCs were collected from male mice carryingEGFP or c-myc-tagged nuclear Akt transgene under the control of α-MHCpromoter (FIG. 12A). Thus, the destiny of BMPCs within the recipientheart was determined by genetic tagging with EGFP, cell fate trackingwith EGFP and c-myc, cell genotyping by sex-chr identification (FIGS.12B and C), EGFP and c-mye gene detection by PCR, mRNA transcripts forEGFP and c-myc-tag by RT-PCR, and protein expression for EGFP andc-myc-tag by Western blotting. Additionally, a critical part of theexperiments was the development of a methodology in which primaryantibodies are directly labeled with quantum dots (36, 139, 207). Thisprotocol eliminates the need for secondary antibody and avoids theinterference of autofluorescence in the specificity of the reaction(57).

The results show that BMPCs integrate within the host heart where theyestablish temporary niches which create the microenvironment necessaryfor the engrafted cells to acquire the cardiac fate and form de novomyocardium (FIG. 13; 57). These data are consistent with our hypothesisand may offer mechanistic insights on the positive results recentlyobtained in double blind clinical trials (208, 209). These findingssuggest that myocardial regeneration is a likely possibility and BMPCsmay have implications for the treatment of the transplanted heart.

B. Cell Fusion

In several studies of myocardial regeneration, we have never foundexamples of fusion between BMPCs or other progenitor cells with residentcardiac cells (47, 48, 51, 59, 137, 139). We have investigated thepossibility of cell fusion in several conditions by measuring the numberof sex chromosomes in newly formed cardiomyocytes and coronary vessels(14, 47, 51, 57, 139, 217). For example, with this approach, we haveexcluded that male myocytes and vessels present in female transplantedhearts are the product of cell fusion (see FIG. 2). Additionally, wehave employed the Cre-Lox genetic system to evaluate whether human MPCscan form human myocardium within the infarcted mouse or rat heart (139)and whether myocardial regeneration in these models is, at least inpart, the product of fusion events. We have found no indication thatcell fusion contributes to cardiac repair (FIG. 14). To exclude thepossibility that heterokaryons are formed when the donor hear iscolonized with recipient progenitor cells, the number of sex chromosomesis measured in the newly formed structures to assess the participationof cell fusion in myocardial regeneration.

C. Paracrine Effects

We test the possibility of a paracrine effect of administered progenitorcells by giving BrdU chronically after transplantation by a wellestablished protocol (51, 57, 139). Over time, cumulative BrdU labelingof female myocytes and coronary vessels provides a quantitativemeasurement of the cellular responses of the donor heart to theinjection of recipient male progenitor cells. Additionally, the fractionof cycling female myocytes, ECs and SMCs at sacrifice is determined byKi67, MCNM5 and phospho-H3 labeling to assess the characteristics of thedonor myocardium at the end of the study. Finally, the number of femalemyocytes and vessels is determined to define the composition of thedonor heart and its changes with time. The injected cells could alsoattenuate cell death mechanisms. Thus, apoptosis of EGFP-, β-Gal- orRFP-negative and Y-chr negative cells is measured. Similarly, thecontribution of male myocytes and vessels to the restoration ofimmunocompatible structures within the non-immunocompatible myocardiumis evaluated.

D. Implantation of BMPCs in Transplanted Donor Heart in Canines (1) CellPreparation

The bone marrow is harvested from the iliac crests through a Jamshidineedle (13G×2″) (218); ˜10 ml of bone marrow is obtained. This protocolyields a total of 20×10⁸ mononuclear cells in each recipient dog.Following lysis of red blood cells in NH4Cl/K, cells are enriched byequilibrium centrifugation over a cushion of Ficoll-Hypaque-400 at adensity of 1.077 g/ml. For lineage-depletion, mononuclear cells areincubated with immunomagnetic beads conjugated with monoclonalantibodies for CD3 (T lymphocytes), CD20 (B lymphocytes), CD33 (myeloidprogenitors), CD14 and CD15 (monocytes). The lineage-negative fractionis exposed to c-kit-conjugated-immunobeads (clone AC126). A smallaliquot is analyzed by FACS to confirm the purity of the preparation(47, 168). Cells are stained with c-kit-A3C6E2 antibody that does notcross-react with the epitope recognized by the AC126 antibody; ˜10×10⁶c-kit-positive BMPCs are collected from each recipient dog. Since atmost 5 injections of BMPCs is done in each dog, 2×10⁶ cells are injectedeach time.

(2) Intervals

One month after transplantation, dogs are anesthetized for left heartcatheterization and cell injection (Group 1; EGFP-labeled cells). Thisprotocol is then applied twice for the next month (Group 2;β-Gal-labeled cells) and subsequently twice for an additional month(Group 3; RFP-labeled cells). Group 1 animals are sacrificed one monthafter a single cell injection, 2 months after transplantation; Group 2animals are sacrificed one month after the last of 3 cell injections, 3months after transplantation; and Group 3 animals are sacrificed onemonth after the last of 5 cell injections, 4 months aftertransplantation. Another group of animals, Group 4, is injectedbi-weekly for 2 months (5 injections) with BMPCs infected with threelentiviruses carrying reporter genes driven by cell lineage specificpromoters. These animals are sacrificed 4 months after transplantationmimicking Group 3. The end-points indicated here are formulated toevaluate the progressive accumulation of immunocomnpatible myocardium.In Groups 1-3, BrdU is injected twice a day (50 mg/kg b.w.×2) to labelforming cells over time (64).

(3) Immunocompatible Myocytes and Coronary Vessels

Three methods are used to detect immunocompatible myocytes and vesselswithin the donor heart: a) Genetic tagging/clonal marking; b) Real-timeRT-PCR and Western blotting for reporter genes; and c) Structuralanalysis. Because of these objectives, at sacrifice the donor heart issubdivided into two parts: one for genetic tagging and biochemicalanalysis of BMPC transdifferentiation and the second for thequantitative characterization of the contribution of donor andregenerated recipient myocardium to the transplanted heart.

a) Genetic tagging/clonal marking (see Example 4): The objective is todocument whether the site of integration of the EGFP, β-gal and RFPlentivirus in BMPCs is found in the committed progeny. This demonstratesthat BMPCs transdifferentiate and have the ability to form de novomyocardium.

b) Real-time RT-PCR and Western blotting for reporter genes (see Example4).

c) Structural analysis (see Example 3): The morphomnetric approachdeveloped in our laboratory allows us to measure the proportion of newlyformed male myocardial structures and resident female myocardium.

The results of these experiments in canines is expected to show thatBMPCs isolated from the recipient male dog will differentiate intomyocytes, smooth muscle cells, and endothelial cells and generateimmunocompatible myocardium and myocardial vessels in the female donorheart. No evidence of fusion events between the male bone marrowprogenitor cells and the donor female myocardial cells is expected to beobserved.

Example 3 Implantation of Vascular Progenitor Cells from the Recipientinto the Transplanted Donor Heart Generates Immunocompatible CoronaryVessels Improving the Evolution of the Cardiac Graft

Cardiac allograft vasculopathy (CAV) is a major pathological event whichseverely affects the unfavorable evolution of the transplanted heart(72, 85, 91-93, 108, 109). In this Example, the therapeutic potential ofvascular progenitor cells (VPCs) is defined. This cell category is themost powerful for the replacement of the coronary circulation of thedonor heart with immunocompatible vessels and thus, the possibility tointroduce stem cell therapy for the treatment of coronary artery diseaseis dramatically advanced. Moreover, the documentation that progenitorcells with angiogenic properties reside in the heart questions thenotion that the bone marrow is the exclusive reservoir or source of stemcells for therapeutic vasculogenesis and points to VPCs as the cell ofchoice for biological bypass. Two issues are addressed in this Example:cell engraftment and necessity to create the various portions of thecoronary circulation.

A. Engraftment

The criteria that govern myocardial regeneration and replacement ofdamaged non-immunocompatible tissue with functionally competentimmunocompatible myocardium involve the ability of the deliveredprogenitor cells to home to or in proximity of the injured sitestogether with the permissive behavior of the donor myocardium (47, 48,50-53, 59, 137, 139, 173, 221). These variables dictate the number ofcells that actually engraft within the hostile environment of the targetorgan which, in turn, condition the efficacy of cell therapy.Engraitment necessitates a surrounding where the cells can survive,divide and differentiate (64, 163, 165-167). Based on data with BMPCs(57) and treated transplanted hearts (FIG. 15), cell engraftment iscompleted in a few days. Junctional and adhesion proteins are present onthe implanted cells documenting a successful interaction betweenprogenitor cells and resident cells; connexin 43 and 45, and N- andE-cadherin have been found between progenitor cells and myocytes orfibroblasts which operate as supporting cells (57, 137, 169). Also,apoptosis occurs in non-engrafted cells and cell division occurs inengrafted cells (FIG. 17). Thus, the destiny of VPCs is established bymeasuring cell death and proliferation and their integration withresident cardiac cells 2-5 days after injection.

B. Coronary Vasculature

The identification and characterization of a coronary VPC discussedabove raises the possibility that the heart harbors a coronary VPC whichregulates the turnover, growth and differentiation of the coronarycirculation. To test this possibility, human VPCs were infected with alentivirus expressing EGFP and were subsequently examined for theability to create functionally competent coronary vessels in dogs withcritical coronary artery stenosis. These VPCs formed human conductiveand intermediate-sized coronary arteries together with small resistancearterioles and capillary profiles within the immunosuppressed recipientmyocardium, restoring in part myocardial blood flow to the distalportion of the heart (FIG. 16). Thus, VPCs may be implemented for thereplacement of coronary vessels affected by allograft vasculopathy inthe transplanted dog heart.

As described in Example 2 with BMPCs, at most 5 injections of VPCs aredone in each dog; 2×10⁶ cells are injected each time.

C. Analysis of the Transplanted Heart

At sacrifice the donor heart is subdivided into two parts: one for theidentification by genetic tagging/clonal marking andbiochemical-molecular parameters of progenitor cell differentiation incardiac lineages (see Example 4) and the second for the quantitativecharacterization of the contribution of donor and regenerated recipientmyocardium to the transplanted heart discussed below.

The first portion of the heart is enzymatically digested (see Example4). The second portion of the heart is utilized for the evaluation ofmyocytes and coronary vessels of donor and recipient origin. A branch ofthe coronary artery is cannulated and the heart and coronary vasculatureis fixed by perfusion with formalin. Briefly, the chest is opened,20,000 units of heparin is given intravenously, and vessels originatingfrom the aortic arch is ligated. The heart is arrested in diastole, thedescending aorta ligated and cannulated. The heart is perfused withphosphate buffer and before perfusion with formalin, a portion of theheart is removed for cell isolation and the studies discussed in Example4 (64, 232-237). The expression of EGFP, β-gal or RFP together with thelocalization of the Y-chromosome allows us to distinguish structuresmediated by growth and differentiation of progenitor cells at differenttime points following cardiac transplantation. In contrast,cardiomyocytes and coronary vessels negative for these reporter proteinsand showing the female genotype constitute the remaining component ofthe donor myocardium.

D. Cell Engraftment

The ability of male EGFP-, β-gal- and RFP-tagged VPCs to home and formjunctional complexes with resident female cardiac cells is determined aspreviously performed (57, 137, 139, 169). This analysis includes theidentification of connexin 43, connexin 45, N-cadherin, E-cadherin, andL-selectin. The number of actually engrafted cells is measuredquantitatively (57).

E. Cell Growth and Death

The progeny of the injected cells is recognized by the presence of EGFP,β-gal and RFP. This labeling is combined with the detection of BrdUindicative of newly formed cells and cumulative growth (169). Thismeasurement is complemented by the expression of MCM5, Ki67 andphospho-H3 to recognize the fraction of cycling cells (MCM5, Ki67) andin mitosis (phospho-H3). The degree of apoptosis and necrosis in VPCsand their progeny is also measured (139, 169, 232-237).

F. Coronary Vasculature and Cardiomyocytes

Morphometric measurements of coronary vessels require a specificapproach. This technique and its theoretical principles have beenpreviously described (136). This protocol applies to the analysis ofcapillaries as well (40, 47, 48, 59). Classes of vessels positive forEGFP, β-gal or RFP together with the Y-chromosome is measuredseparately. An identical analysis is conducted in vessels which arenegative for these markers. Thus, an estimation of the immunocompatibleand non-immunocompatible coronary vasculature is obtained.

The number of newly formed and existing myocytes is measuredquantitatively by a protocol developed previously which includesmeasurements in tissue sections and isolated cells (136). Sampling forcoronary circulation and cardiomyocytes: see ref. 136.

The results of these experiments are expected to show that vascularprogenitor cells isolated from the explanted heart of the male recipientdog will engraft in the donor female heart after administration anddifferentiate into predominantly smooth muscle cells and endothelialcells. The differentiated cells will assemble into immunocompatiblecoronary vasculature (coronary arteries, arterioles, and capillaries)similar to that formed after the administration of human vascularprogenitor cells (see section B above).

Example 4 Implantation of MPCs from the Recipient into the TransplantedDonor Heart Generates Immunocompatible Cardiomyocytes Improving theEvolution of the Cardiac Graft

Myocyte progenitor cells (MPCs) are programmed to give rise tocardiomyocytes and these cells should be superior to bone marrowprogenitor cells (BMPCs) and vascular progenitor cells (VPCs) forreplacement of lost muscle mass. This is suggested by in vitro resultsin which the differentiated progenies of MPCs and VPCs were compared(see FIG. 7). BMPCs differentiate into myocytes (239-241), smooth musclecells (242, 243) and endothelial cells (244, 245) in vitro. However, therelative proportion of these cell types in the same preparation isdifficult to obtain.

To address the issue of the regenerative capacity of the differentprogenitor cell classes (e.g. BMPCs, MPCs, and VPCs), the quantitativemeasurements of newly formed myocytes and vessels based on morphometricprinciples and immunolabeling (Example 3) is complemented with a novelgenetic-molecular assay developed previously in our laboratory (246).Thus, two complementary methods are used: a genetic-molecular assay anda structural assay. Genetic tagging is discussed below together with aseries of other molecular determinations.

A lentivirus expressing EGFP, P-gal and RFP is employed for in vitroinfection of BMPCs, VPCs and MPCs. The detection of the lentiviralintegration site is based on the premise that it contains tworestriction enzyme (RE) cleavage sites at a reasonable distance (50-2000bp) from the lentiviral LTRs located at the 3′ and 5′ sites of the viralgenome. Following the cleavage of the genomic DNA with the RE, DNAproducts are self-ligated to produce circularized DNA. This step createsa genomic sequence of a length that is variable in view of the randomlocation of the RE site within the region of the dog genome flanking theviral DNA. The unknown lentiviral flanking region is entrapped betweentwo known sequences and can be, therefore, amplified by PCR and resolvedon gel; each band corresponds to one insertion site. Data have beenobtained after intramyocardial injection of humran cardiac progenitorcells in immunosuppressed infarcted rats to validate this novel approach(246; FIG. 17).

The data indicate that similarities exist between forming myocardium(36, 51, 57, 59, 64, 139) and late-fetal and postnatal cardiacmaturation (136, 247, 248). The volume of myocytes is comparablealthough differences in number exist; regeneration tends to recapitulatethe processes present in the fetal-neonatal heart (249, 250).Accordingly, the prenatal and postnatal heart are used for comparison.

Hearts from female donors are transplanted into male recipients asdescribed in Example 1. Progenitor cells isolated from the maleexplanted heart are infected with lentivirus carrying EGFP, β-gal andRFP. The infected progenitor cells are then injected into thetransplanted donor heart. At most 5 injections of progenitor cells aredone in each dog; 2×10⁶ cells are injected each time.

Molecular assays and immunocytochemistry are used to identify thetime-course of myocardial regeneration in the transplanted heart and inthe developing heart (fetal dog heart at 40 and 60 days of gestation,1-2 days after birth, at the time of weaning, 4-5 weeks, and 8 months).The expression of transcription factors that control myogenesis andvasculogenesis is determined (247, 248, 251-253). Moreover, membrane andcytoplasmic proteins specific of myocytes, smooth muscle cells andendothelial cells are studied (254-257). Molecular and cytochemicaldetections are both relevant to obtain information with twocomplementary methods and ensure that protein expression is properlydistributed within cells. Postnatally, apoptosis decreases rapidly andthis adaptation is paralleled by a reduction in myocyte formationcoupled with binucleation of the enlarging myocytes (258-260). Myocytekaryokinesis in the absence of cytokinesis is accompanied bydownregulation of the transcription factor Tsc (tuberous sclerosiscomplex) and upregulation of Gax (growth arrest gene). Similaradaptations may occur in the transplanted heart.

Transplanted Heart: At sacrifice, in a portion of the heart, a coronaryartery branch is cannulated and cardiac cells are enzymaticallydissociated with collagenase for biochemical-molecular determinations(232-237). Large myocytes of donor origin and small cardiac cells (newlyformed myocytes and non-myocytes) of both donor and recipient origin areseparated by Ficoll gradient and differential centrifugation (246).Within the small cells, the progeny of the injected progenitor cells aresorted by FACS based on the presence of EGFP, β-gal and RFP. Thespontaneous fluorescence of EGFP- and RFP-positive cells allow theirdirect collection. When β-gal-positive progenitor cells are injected,the isolated cells are fixed in 4% paraformaldehyde for 15 min, stainedwith anti β-gal antibody (168) and sorted by FACS. Cells are employeddirectly for RNA extraction and real-time RT-PCR. For genetic tagging,an additional step is required. Progenitor cells are sorted by FACS onthe basis of c-kit and flk1 expression (47, 59, 64, 139). C-kit-negativecells are subdivided in CD31-positive (endothelial cells),calponin-positive (smooth muscle cells), ac-SA-positive (myocytes) andprocollagen-I-positive (fibroblasts) cells.

Genetic tagging: Genomic DNA is extracted and employed for the detectionof the site of viral integration. Importantly, each cell populationisolated from each heart is processed separately according to the AKANEprotocol (see FIG. 17 and legend). Primers are designed to include aportion of the coding regions of EGFP, β-gal and RFP to distinguish thethree viral genomes. Amplified DNA is run on agarose gel. Bands are cut,DNA extracted and sequenced from both ends to determine the insertionsite of each clone (246).

Biochemical-molecular determinations: These analyses are performed infreshly isolated EGFP-, β-gal- and RFP-positive cells and whole lysatesof fetal, neonatal and young adult hearts by real time RT-PCR (139,263). Western blotting is also employed. These analyses are complementedby the detection of proteins by immnunocytochemistry. For real-timeRT-PCR, total RNA is extracted with Trizol or RecoverAll™ Total NucleicAcid Isolation Ambion Kit which is designed forformalin/paraformaldehyde fixed structures (139, 263).

Changes in expression of transcription factors involved indifferentiation of myocytes (Nkx2.5, GATA4, MEF2C, Tbx, SRF, HAND-1,HAND-2), smooth muscle cells (GATA6) and endothelial cells (Ets1, Vezf1)are determined. Moreover, mRNA expression of membrane and cytoplasmiccomponents specific of myocytes (connexin 43, N-cadherin, troponin I,atrial and ventricular MLC-2, α- and β-MHC, α-SA), smooth muscle cells(α-SMA, TGF-βR) and endothelial cells (eNOS, CD31, vWf) is studied. Formnyocyte karyokinesis and cytokinesis, Tsc and Gax expression isdetermined.

PCR for Y-chromosome DNA: Primers are employed to detect Sry, the sexdetermining region of the Y-chromosome: dogSry-F:5′-CGTTGGAACGGACAATTCAACCTCGAA-3 SEQ ID NO.: 1 (26 nt, Tm 61° C.) anddogSry-R: 5′-ACCTGCTTCATAGCATGGAGGAGGA-3′ SEQ ID NO.: 2 (26 nt, Tm 64°C.) [amplicon size: 369 bp].

Ilmmunocytochemistry: These analyses are conducted by confocalmicroscopy of isolated cells and developing heart to complement thereal-time RT-PCR studies.

The results of this set of experiments is expected to show the myocyteprogenitor cells will generate predominantly new myocytes of recipientorigin, while vascular progenitor cells will generate predominantlysmooth muscle cells and endothelial cells of recipient origin. The bonemarrow progenitor cells will generate myocytes, smooth muscle cells, andendothelial cells of recipient origin, but are expected to generatefewer numbers of myocytes than myocyte progenitor cells. It is alsoexpected that the generation of immunocompatible myocardium by theimplanted progenitor cells will be comparable to the formation ofmyocardial tissue during development.

Example 5 Implantation of VIPCs and MPCs into the Transplanted DonorHeart Generates Immunocompatible Coronary Vessels and Cardiomyocyteswhich Together Reconstitute an Immunocompatible Heart

The objective of this Example is to utilize therapeutically the tworecently identified progenitor cell classes, vascular progenitor cells(VPCs) and myocyte progenitor cells (MPCs), to replace donor myocardiumwith new recipient myocardium. This approach takes advantage of thevessel regenerative capacity of VPCs and myocyte formation of MPCs todramatically restructure the transplanted heart. Human studies oncardiac chimerism (14-26) are consistent with this possibility and, infact, point strongly in this direction. The premise is that residentMPCs and VPCs are preferable and more efficient in creating de novomyocardium than bone marrow progenitor cells (BMPCs) which have totransdifferentiate and acquire a different genetic phenotype (36, 185,186, 207) before committing to the myocyte and vascular cell lineages(47-57). However, progenitor cells from the explanted organ and/or thebone marrow can be employed to rebuild the donor heart.

Three protocols are used to assess whether the implanted progenitorcells generate functional myocardial tissue and vessels: regionalventricular function, coronary blood flow, and myocyte mechanics.

A. Ventricular Function

The use of a large animal model offers the unique opportunity toinstrument the transplanted heart with sonomicrometers and determinechronically the time course of the alterations in regional function andestablish whether delivery of progenitor classes results in animprovement of the dyskinetic and hypokinetic segments included withinthe sonomicrometers (64, 232-237). Recovery of contraction points tomyocardial regeneration as one of the possible mechanisms involved,while the lack of amelioration in function suggests absence of tissuereconstitution. The histological examination of the same regions atsacrifice allows us to obtain critical information on the structure andfunction of the transplanted heart.

B. Coronary Blood Flow (CBF) and Hemodynamics

The potential efficacy of cell therapy requires the inclusion ofmeasurements of CBF distribution and coronary vascular resistance(264-267). If the formation of the various segments of the coronarycirculation by BMPCs, VPCs and/or MPCs occurs and cardiac allograftvasculopathy (CAV) is partly corrected, a functional counterpart has tobe documented. The collateral circulation of the canine heart protectsto a certain extent the myocardium from ischemic events (264-267) sothat coronary occlusion may result in hypokinesis instead of dyskinesisof the affected region of the heart (64). Therefore, the potentiation ofcollateral vessel formation has to be considered and carefully analyzedtogether with angiogenesis and vasculogenesis to interpret properlyfunctional changes in CBF (FIG. 18). Data are obtained at days aftereach cell treatment and at sacrifice (264, 265, 268-270).

C. Myocyte Mechanics

An important aspect of the reconstituting myocardium concerns theperformance of regenerated myocytes. Thus, parameters of myocytecontractility, calcium handling and L-type calcium current are measuredin isolated myocytes and comparisons are made with donor myocytes (40,57, 59, 139, 271). The acquisition of this infbrmation allows us toestablish the effective functional competence of regenerated cells (59,139). Changes in myocyte mechanics which accompany the acquisition ofthe adult phenotype are assessed and analyzed in myocyte populationsderived from different progenitor classes. Thus, the most efficient andpowerful myocyte progeny are identified. These measurements areroutinely performed (40, 57, 59, 139, 271).

D. Specific Methods

Intervals: As discussed in Example 2, during the course of the study,dogs are anesthetized for left heart catheterization and autologousprogenitor cell injection (Group 1; 50% EGFP-labeled VPCs and 50%RFP-labeled MPCs). This protocol is then applied bi-weekly for the nextmonth (Group 2; 50% EGFP-labeled VPCs and 50% β-Gal-labeled MPCs) andsubsequently for an additional month (Group 3; 50% EGFP-labeled VPCs and50% β-Gal-labeled MPCs). Group 1 animals are sacrificed one month aftera single cell injection, 2 months after transplantation; Group 2 animalsare sacrificed one month after the last of 3 cell injections, 3 monthsafter transplantation; and Group 3 animals are sacrificed one monthafter the last of 5 cell injections, 4 months after transplantation.Another group of animals, Group 4, is injected bi-weekly for 2 months (5injections) with 50% MPCs infected with a lentivirus carryingEGFP-Flag-tag under the cardiornyocyte specific α-MHC promoter,RFP-HA-tag under the SMC-specific Sm22a promoter and TFP-c-myc-tag underthe EC-specific VE-cadherin promoter. These animals are sacrificed 4months after transplantation. At most 5 injections of progenitor cellsare done in each dog; 2×10⁶ cells are injected each time.

CBF: Two ml of Steri spheres (BioPal) suspension (2 millions/mL) aremixed with arterial blood and injected into left atrium over 5 seconds.Immediately before the injection, arterial blood reference sample iswithdrawn from the aortic catheter. At the end of experiment, transmuraltissue samples (˜1 g) are harvested from cardiac regions of interest andcut into three layers: epicardial, mid-myocardial and endocardial.Tissue and reference blood samples are dried overnight, and then shippedto Bio-Pal for neutron activation and radioactivity counting. Theaverage radioactivity counts for myocardium are calculated asCi=(C1×W1+C2×W2+C3×W3)/(W1+W2+W3) where Ci is the average count (dpm/g),C1-3 and W1-3 are the counts and wet weights for the epicardium,mid-myocardium, and endocardium layers, respectively. The myocardialflow is calculated as Qi=(Ci/Cref)×R(ml/min) where Qi is flow, Ci andCref are the radioactivities in tissue and in blood reference sample,respectively, and R is the withdrawal rate of the reference bloodsample. Endocardial/epicardial flow ratio is calculated to obtain anindex of regional flow pattern (268-270).

Mechanics, Ca2+ transients and electrophysiology (see FIG. 19): Myocytesare transferred to a chamber placed on the stage of an invertedmicroscope. External bath Ca2+ is kept at 1.5 mM. Mechanics, Ca2+transients: Myocytes are stimulated at 1.0 Hz by rectangulardepolarizing pulses, 2 ms in duration, and twice-diastolic thresholdintensity. Changes in cell length are quantified by edge tracking.Sarcomere length is determined by the mean frequency of sarcomerespacing utilizing the Fast Fourier Transform. Fluo 3-fluorescence ismeasured by epi-illumination with flashes of 488 nm light. After loadingcell with the Ca2+ probe, experiments are performed at 25±0.2° C. tominimize the loss of the Ca2+ indicator. The ability of myocytes toadapt to different rates of stimulation and extracellular Ca2+concentrations is examined (40, 59). Electrophysiology: Electricalproperties of differentiating myocytes are measured in combination withcell shortening. Data are collected by whole cell patch-clamp techniquein voltage- and current-clamp mode and by edge motion detectionmeasurements. Voltage, time-dependence and density of L-type Ca2+current is analyzed in voltage-clamp preparations. T-type Ca2+ currentis assessed (265); it is restricted to developing myocytes (272).Relationship between shortening and action potential is done incurrent-clamp mode (57, 271, 273-280).

The results of these experiments are expected to show that the implantedMPCs will generate predominantly immunocompatible myocytes, while theimnplanted VPCs will generate predominantly immnnocompatible smoothmuscle cells and endothelial cells. Some of the smooth muscle cells andendothelial cells derived from the recipient will assemble intofunctional coronary vessels that may reduce cardiac allograftvasculopathy and enhance coronary blood flow.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theappended claims is not to be limited by particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope thereof.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

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1-11. (canceled)
 12. A method of reducing an immune response to atransplanted donor heart in a recipient subject comprising:administering isolated adult bone marrow progenitor cells to thetransplanted donor heart, wherein said adult bone marrow progenitorcells are isolated from the recipient subject's bone marrow and arelineage negative and c-kit positive, wherein said bone marrow progenitorcells generate immunocompatible myocardium and immunocompatiblemyocardial vessels following their administration, thereby reducing theimmune response to said transplanted donor heart.
 13. (canceled)
 14. Themethod of claim 12, wherein said bone marrow progenitor cellsdifferentiate into immunocompatible endothelial cells, smooth musclecells, and cardiomyocytes.
 15. The method of claim 12, wherein said bonemarrow progenitor cells are activated by exposing the cells to one ormore cytokines prior to administration.
 16. The method of claim 12,wherein said bone marrow progenitor cells are administered immediatelyafter transplantation.
 17. The method of claim 12, further comprising:administering isolated adult cardiac progenitor cells to thetransplanted donor heart, wherein said adult cardiac progenitor cellsare isolated from the recipient subject's myocardial tissue and arelineage negative and c-kit positive.
 18. The method of claim 17, whereinsaid isolated adult cardiac progenitor cells are administered bymultiple administrations after transplantation.
 19. The method of claim18, wherein said multiple administrations occur at a set interval afteradministration of said bone marrow progenitor cells.
 20. The method ofclaim 12, further comprising administering to the subject animmunosuppressive therapy. 21-28. (canceled)
 29. The method of claim 17,wherein prior to administration, said cardiac progenitor cells areseparated into vascular progenitor cells and myocyte progenitor cells,wherein said vascular progenitor cells and said myocyte progenitor cellsare each expanded in culture to obtain pure cultures of each of saidvascular progenitor cells and said myocyte progenitor cells, and eachare subsequently administered to said transplanted donor heart, whereinsaid vascular progenitor cells are vascular endothelial growth factorreceptor 2 (VEGFR2) positive and differentiate into immunocompatibleendothelial cells and smooth muscle cells, and wherein said myocyteprogenitor cells are VEGFR2 negative and differentiate intoimmunocompatible cardiomyocytes.
 30. The method of claim 12, whereinsaid subject is human.
 31. The method of claim 12, wherein said adultbone marrow progenitor cells are expanded in culture prior toadministration to the donor heart.
 32. The method of claim 12, whereinsaid adult bone marrow progenitor cells are administered to thetransplanted donor heart by intramyocardial or intracoronary injection.